Heterocyclic compounds and their uses

ABSTRACT

Substituted bicyclic heteroaryls and compositions containing them, for the treatment of general inflammation, arthritis, rheumatic diseases, osteoarthritis, inflammatory bowel disorders, inflammatory eye disorders, inflammatory or unstable bladder disorders, psoriasis, skin complaints with inflammatory components, chronic inflammatory conditions, including but not restricted to autoimmune diseases such as systemic lupus erythematosis (SLE), myestenia gravis, rheumatoid arthritis, acute disseminated encephalomyelitis, idiopathic thrombocytopenic purpura, multiples sclerosis, Sjoegren&#39;s syndrome and autoimmune hemolytic anemia, allergic conditions including all forms of hypersensitivity, The present invention also enables methods for treating cancers that are mediated, dependent on or associated with p110δ activity, including but not restricted to leukemias, such as Acute Myeloid leukaemia (AML) Myelo-dysplastic syndrome (MDS) myelo-proliferative diseases (MPD) Chronic Myeloid Leukemia (CML) T-cell Acute Lymphoblastic leukaemia (T-ALL) B-cell Acute Lymphoblastic leukaemia (B-ALL) Non Hodgkins Lymphoma (NHL) B-cell lymphoma and solid tumors, such as breast cancer.

This application claims the benefit of U.S. Provisional Application No.61/620,270, filed Apr. 4, 2012, which is hereby incorporated byreference.

The present invention relates generally to phosphatidylinositol 3-kinase(PI3K) enzymes, and more particularly to selective inhibitors of PI3Kactivity and to methods of using such materials.

BACKGROUND OF THE INVENTION

Cell signaling via 3′-phosphorylated phosphoinositides has beenimplicated in a variety of cellular processes, e.g., malignanttransformation, growth factor signaling, inflammation, and immunity (seeRameh et al., J. Biol Chem, 274:8347-8350 (1999) for a review). Theenzyme responsible for generating these phosphorylated signalingproducts, phosphatidylinositol 3-kinase (PI 3-kinase; PI3K), wasoriginally identified as an activity associated with viral oncoproteinsand growth factor receptor tyrosine kinases that phosphorylatesphosphatidylinositol (PI) and its phosphorylated derivatives at the3′-hydroxyl of the inositol ring (Panayotou et al., Trends Cell Biol2:358-60 (1992)).

The levels of phosphatidylinositol-3,4,5-triphosphate (PIP3), theprimary product of PI 3-kinase activation, increase upon treatment ofcells with a variety of stimuli. This includes signaling throughreceptors for the majority of growth factors and many inflammatorystimuli, hormones, neurotransmitters and antigens, and thus theactivation of PI3Ks represents one, if not the most prevalent, signaltransduction events associated with mammalian cell surface receptoractivation (Cantley, Science 296:1655-1657 (2002); Vanhaesebroeck et al.Annu Rev. Biochem, 70: 535-602 (2001)). PI 3-kinase activation,therefore, is involved in a wide range of cellular responses includingcell growth, migration, differentiation, and apoptosis (Parker et al.,Current Biology, 5:577-99 (1995); Yao et al., Science, 267:2003-05(1995)). Though the downstream targets of phosphorylated lipidsgenerated following PI 3-kinase activation have not been fullycharacterized, it is known that pleckstrin-homology (PH) domain- andFYVE-finger domain-containing proteins are activated when binding tovarious phosphatidylinositol lipids (Sternmark et al., J Cell Sci,112:4175-83 (1999); Lemmon et al., Trends Cell Biol, 7:237-42 (1997)).Two groups of PH-domain containing PI3K effectors have been studied inthe context of immune cell signaling, members of the tyrosine kinase TECfamily and the serine/threonine kinases of to AGC family. Members of theTec family containing PH domains with apparent selectivity for PtdIns(3,4,5)P₃ include Tec, Btk, Itk and Etk. Binding of PH to PIP₃ iscritical for tyrsosine kinase activity of the Tec family members(Schaeffer and Schwartzberg, Curr. Opin. Immunol. 12: 282-288 (2000))AGC family members that are regulated by PI3K include thephosphoinositide-dependent kinase (PDK1), AKT (also termed PKB) andcertain isoforms of protein kinase C (PKC) and S6 kinase. There arethree isoforms of AKT and activation of AKT is strongly associated withPI3K-dependent proliferation and survival signals. Activation of AKTdepends on phosphorylation by PDK1, which also has a3-phosphoinositide-selective PH domain to recruit it to the membranewhere it interacts with AKT. Other important PDK1 substrates are PKC andS6 kinase (Deane and Fruman, Annu Rev. Immunol. 22_(—)563-598 (2004)).In vitro, some isoforms of protein kinase C (PKC) are directly activatedby PIP3. (Burgering et al., Nature, 376:599-602 (1995)).

Presently, the PI 3-kinase enzyme family has been divided into threeclasses based on their substrate specificities. Class I PI3Ks canphosphorylate phosphatidylinositol (PI),phosphatidylinositol-4-phosphate, andphosphatidyl-inositol-4,5-biphosphate (PIP2) to producephosphatidylinositol-3-phosphate (PIP),phosphatidylinositol-3,4-biphosphate, andphosphatidylinositol-3,4,5-triphosphate, respectively. Class II PI3Ksphosphorylate PI and phosphatidyl-inositol-4-phosphate, whereas ClassIII PI3Ks can only phosphorylate PI.

The initial purification and molecular cloning of PI 3-kinase revealedthat it was a heterodimer consisting of p85 and p110 subunits (Otsu etal., Cell, 65:91-104 (1991); Hiles et al., Cell, 70:419-29 (1992)).Since then, four distinct Class I PI3Ks have been identified, designatedPI3K α, β, δ, and γ, each consisting of a distinct 110 kDa catalyticsubunit and a regulatory subunit. More specifically, three of thecatalytic subunits, i.e., p110α, p110β and p110δ, each interact with thesame regulatory subunit, p85; whereas p110γ interacts with a distinctregulatory subunit, p101. As described below, the patterns of expressionof each of these PI3Ks in human cells and tissues are also distinct.Though a wealth of information has been accumulated in recent past onthe cellular functions of PI 3-kinases in general, the roles played bythe individual isoforms are not fully understood.

Cloning of bovine p110α has been described. This protein was identifiedas related to the Saccharomyces cerevisiae protein: Vps34p, a proteininvolved in vacuolar protein processing. The recombinant p110α productwas also shown to associate with p85α, to yield a PI3K activity intransfected COS-1 cells. See Hiles et al., Cell, 70, 419-29 (1992).

The cloning of a second human p110 isoform, designated p110β, isdescribed in Hu et al., Mol Cell Biol, 13:7677-88 (1993). This isoformis said to associate with p85 in cells, and to be ubiquitouslyexpressed, as p110β mRNA has been found in numerous human and mousetissues as well as in human umbilical vein endothelial cells, Jurkathuman leukemic T cells, 293 human embryonic kidney cells, mouse 3T3fibroblasts, HeLa cells, and NBT2 rat bladder carcinoma cells. Such wideexpression suggests that this isoform is broadly important in signalingpathways.

Identification of the p110δ isoform of PI 3-kinase is described inChantry et al., J Biol Chem, 272:19236-41 (1997). It was observed thatthe human p110δ isoform is expressed in a tissue-restricted fashion. Itis expressed at high levels in lymphocytes and lymphoid tissues and hasbeen shown to play a key role in PI 3-kinase-mediated signaling in theimmune system (Al-Alwan etl al. JI 178: 2328-2335 (2007); Okkenhaug etal JI, 177: 5122-5128 (2006); Lee et al. PNAS, 103: 1289-1294 (2006)).P110δ has also been shown to be expressed at lower levels in breastcells, melanocytes and endothelial cells (Vogt et al. Virology, 344:131-138 (2006) and has since been implicated in conferring selectivemigratory properties to breast cancer cells (Sawyer et al. Cancer Res.63:1667-1675 (2003)). Details concerning the P1106 isoform also can befound in U.S. Pat. Nos. 5,858,753; 5,822,910; and 5,985,589. See also,Vanhaesebroeck et al., Proc Nat. Acad Sci USA, 94:4330-5 (1997), andinternational publication WO 97/46688.

In each of the PI3Kα, β, and δ subtypes, the p85 subunit acts tolocalize PI 3-kinase to the plasma membrane by the interaction of itsSH2 domain with phosphorylated tyrosine residues (present in anappropriate sequence context) in target proteins (Rameh et al., Cell,83:821-30 (1995)). Five isoforms of p85 have been identified (p85α,p85β, p55γ, p55α and p50α) encoded by three genes. Alternativetranscripts of Pik3r1 gene encode the p85α, p55α and p50α proteins(Deane and Fruman, Annu Rev. Immunol. 22: 563-598 (2004)). p85α isubiquitously expressed while p85β, is primarily found in the brain andlymphoid tissues (Volinia et al., Oncogene, 7:789-93 (1992)).Association of the p85 subunit to the PI 3-kinase p110α, β, or δcatalytic subunits appears to be required for the catalytic activity andstability of these enzymes. In addition, the binding of Ras proteinsalso upregulates PI 3-kinase activity.

The cloning of p110γ revealed still further complexity within the PI3Kfamily of enzymes (Stoyanov et al., Science, 269:690-93 (1995)). Thep110γ isoform is closely related to p110α and p110β (45-48% identity inthe catalytic domain), but as noted does not make use of p85 as atargeting subunit. Instead, p110γ binds a p101 regulatory subunit thatalso binds to the βγ subunits of heterotrimeric G proteins. The p101regulatory subunit for PI3 Kgamma was originally cloned in swine, andthe human ortholog identified subsequently (Krugmann et al., J BiolChem, 274:17152-8 (1999)). Interaction between the N-terminal region ofp101 with the N-terminal region of p110γ is known to activate PI3Kγthrough Gβγ. Recently, a p10′-homologue has been identified, p84 orp87^(PIKAP) (PI3Kγ adapter protein of 87 kDa) that binds p110γ (Voigt etal. JBC, 281: 9977-9986 (2006), Suire et al. Curr. Biol. 15: 566-570(2005)). p87^(PIKAP) is homologous to p101 in areas that bind p110γ andGβγ and also mediates activation of p110γ downstream ofG-protein-coupled receptors. Unlike p101, p87^(PIKAP) is highlyexpressed in the heart and may be crucial to PI3Kγ cardiac function.

A constitutively active PI3K polypeptide is described in internationalpublication WO 96/25488. This publication discloses preparation of achimeric fusion protein in which a 102-residue fragment of p85 known asthe inter-SH2 (iSH2) region is fused through a linker region to theN-terminus of murine p110. The p85 iSH2 domain apparently is able toactivate PI3K activity in a manner comparable to intact p85 (Klippel etal., Mol Cell Biol, 14:2675-85 (1994)).

Thus, PI 3-kinases can be defined by their amino acid identity or bytheir activity. Additional members of this growing gene family includemore distantly related lipid and protein kinases including Vps34 TOR1,and TOR2 of Saccharomyces cerevisiae (and their mammalian homologs suchas FRAP and mTOR), the ataxia telangiectasia gene product (ATR) and thecatalytic subunit of DNA-dependent protein kinase (DNA-PK). Seegenerally, Hunter, Cell, 83:1-4 (1995).

PI 3-kinase is also involved in a number of aspects of leukocyteactivation. A p85-associated PI 3-kinase activity has been shown tophysically associate with the cytoplasmic domain of CD28, which is animportant costimulatory molecule for the activation of T-cells inresponse to antigen (Pages et al., Nature, 369:327-29 (1994); Rudd,Immunity, 4:527-34 (1996)). Activation of T cells through CD28 lowersthe threshold for activation by antigen and increases the magnitude andduration of the proliferative response. These effects are linked toincreases in the transcription of a number of genes includinginterleukin-2 (IL2), an important T cell growth factor (Fraser et al.,Science, 251:313-16 (1991)). Mutation of CD28 such that it can no longerinteract with PI 3-kinase leads to a failure to initiate IL2 production,suggesting a critical role for PI 3-kinase in T cell activation.

Specific inhibitors against individual members of a family of enzymesprovide invaluable tools for deciphering functions of each enzyme. Twocompounds, LY294002 and wortmannin, have been widely used as PI 3-kinaseinhibitors. These compounds, however, are nonspecific PI3K inhibitors,as they do not distinguish among the four members of Class I PI3-kinases. For example, the IC₅₀ values of wortmannin against each ofthe various Class I PI 3-kinases are in the range of 1-10 nM. Similarly,the IC₅₀ values for LY294002 against each of these PI 3-kinases is about1 μM (Froman et al., Ann Rev Biochem, 67:481-507 (1998)). Hence, theutility of these compounds in studying the roles of individual Class IPI 3-kinases is limited.

Based on studies using wortmannin, there is evidence that PI 3-kinasefunction also is required for some aspects of leukocyte signalingthrough G-protein coupled receptors (Thelen et al., Proc Natl Acad SciUSA, 91:4960-64 (1994)). Moreover, it has been shown that wortmannin andLY294002 block neutrophil migration and superoxide release. However,inasmuch as these compounds do not distinguish among the variousisoforms of PI3K, it remains unclear from these studies which particularPI3K isoform or isoforms are involved in these phenomena and whatfunctions the different Class I PI3K enzymes perform in both normal anddiseased tissues in general. The co-expression of several PI3K isoformsin most tissues has confounded efforts to segregate the activities ofeach enzyme until recently.

The separation of the activities of the various PI3K isozymes has beenadvanced recently with the development of genetically manipulated micethat allowed the study of isoform-specific knock-out and kinase deadknock-in mice and the development of more selective inhibitors for someof the different isoforms. P110α and p110β knockout mice have beengenerated and are both embryonic lethal and little information can beobtained from these mice regarding the expression and function of p110alpha and beta (Bi et al. Mamm. Genome, 13:169-172 (2002); Bi et al. J.Biol. Chem. 274:10963-10968 (1999)). More recently, p110α kinase deadknock in mice were generated with a single point mutation in the DFGmotif of the ATP binding pocket (p110αD^(933A)) that impairs kinaseactivity but preserves mutant p110α kinase expression. In contrast toknock out mice, the knockin approach preserves signaling complexstoichiometry, scaffold functions and mimics small molecule approachesmore realistically than knock out mice. Similar to the p110α KO mice,p110αD^(933A) homozygous mice are embryonic lethal. However,heterozygous mice are viable and fertile but display severely bluntedsignaling via insulin-receptor substrate (IRS) proteins, key mediatorsof insulin, insulin-like growth factor-1 and leptin action. Defectiveresponsiveness to these hormones leads to hyperinsulinaemia, glucoseintolerance, hyperphagia, increase adiposity and reduced overall growthin heterozygotes (Foukas, et al. Nature, 441: 366-370 (2006)). Thesestudies revealed a defined, non-redundant role for p110α as anintermediate in IGF-1, insulin and leptin signaling that is notsubstituted for by other isoforms. We will have to await the descriptionof the p110β kinase-dead knock in mice to further understand thefunction of this isoform (mice have been made but not yet published;Vanhaesebroeck).

P110γ knock out and kinase-dead knock in mice have both been generatedand overall show similar and mild phenotypes with primary defects inmigration of cells of the innate immune system and a defect in thymicdevelopment of T cells (Li et al. Science, 287: 1046-1049 (2000), Sasakiet al. Science, 287: 1040-1046 (2000), Patrucco et al. Cell, 118:375-387 (2004)).

Similar to p110γ, PI3K delta knock out and kinase-dead knock-in micehave been made and are viable with mild and like phenotypes. Thep110δ^(D910A) mutant knock in mice demonstrated an important role fordelta in B cell development and function, with marginal zone B cells andCD5+ B1 cells nearly undetectable, and B- and T cell antigen receptorsignaling (Clayton et al. J. Exp. Med. 196:753-763 (2002); Okkenhaug etal. Science, 297: 1031-1034 (2002)). The p110δ^(D910A) mice have beenstudied extensively and have elucidated the diverse role that deltaplays in the immune system. T cell dependent and T cell independentimmune responses are severely attenuated in p110δ^(D910A) and secretionof TH1 (INF-γ) and TH2 cytokine (IL-4, IL-5) are impaired (Okkenhaug etal. J. Immunol. 177: 5122-5128 (2006)). A human patient with a mutationin p110δ has also recently been described. A taiwanese boy with aprimary B cell immunodeficiency and a gamma-hypoglobulinemia ofpreviously unknown aetiology presented with a single base-pairsubstitution, m.3256G to A in codon 1021 in exon 24 of p110δ. Thismutation resulted in a mis-sense amino acid substitution (E to K) atcodon 1021, which is located in the highly conserved catalytic domain ofp110δ protein. The patient has no other identified mutations and hisphenotype is consistent with p110δ deficiency in mice as far as studied.(Jou et al. Int. J. Immunogenet. 33: 361-369 (2006)).

Isoform-selective small molecule compounds have been developed withvarying success to all Class I PI3 kinase isoforms (Ito et al. J. Pharm.Exp. Therapeut., 321:1-8 (2007)). Inhibitors to alpha are desirablebecause mutations in p110α have been identified in several solid tumors;for example, an amplification mutation of alpha is associated with 50%of ovarian, cervical, lung and breast cancer and an activation mutationhas been described in more than 50% of bowel and 25% of breast cancers(Hennessy et al. Nature Reviews, 4: 988-1004 (2005)). Yamanouchi hasdeveloped a compound YM-024 that inhibits alpha and delta equi-potentlyand is 8- and 28-fold selective over beta and gamma respectively (Ito etal. J. Pharm. Exp. Therapeut., 321:1-8 (2007)).

P110β is involved in thrombus formation (Jackson et al. Nature Med. 11:507-514 (2005)) and small molecule inhibitors specific for this isoformare thought after for indication involving clotting disorders (TGX-221:0.007 uM on beta; 14-fold selective over delta, and more than 500-foldselective over gamma and alpha) (Ito et al. J. Pharm. Exp. Therapeut.,321:1-8 (2007)).

Selective compounds to p110γ are being developed by several groups asimmunosuppressive agents for autoimmune disease (Rueckle et al. NatureReviews, 5: 903-918 (2006)). Of note, AS 605240 has been shown to beefficacious in a mouse model of rheumatoid arthritis (Camps et al.Nature Medicine, 11: 936-943 (2005)) and to delay onset of disease in amodel of systemic lupus erythematosis (Barber et al. Nature Medicine,11: 933-935 (205)).

Delta-selective inhibitors have also been described recently. The mostselective compounds include the quinazolinone purine inhibitors (PIK39and IC87114). IC87114 inhibits p110δ in the high nanomolar range (tripledigit) and has greater than 100-fold selectivity against p110α, is 52fold selective against p110β but lacks selectivity against p110γ(approx. 8-fold). It shows no activity against any protein kinasestested (Knight et al. Cell, 125: 733-747 (2006)). Using delta-selectivecompounds or genetically manipulated mice (p110δ^(D910A)) it was shownthat in addition to playing a key role in B and T cell activation, deltais also partially involved in neutrophil migration and primed neutrophilrespiratory burst and leads to a partial block of antigen-IgE mediatedmast cell degranulation (Condliffe et al. Blood, 106: 1432-1440 (2005);Ali et al. Nature, 431: 1007-1011 (2002)). Hence p110δ is emerging as animportant mediator of many key inflammatory responses that are alsoknown to participate in aberrant inflammatory conditions, including butnot limited to autoimmune disease and allergy. To support this notion,there is a growing body of p110δ target validation data derived fromstudies using both genetic tools and pharmacologic agents. Thus, usingthe delta-selective compound IC 87114 and the p110δ^(D910A) mice, Ali etal. (Nature, 431: 1007-1011 (2002)) have demonstrated that delta plays acritical role in a murine model of allergic disease. In the absence offunctional delta, passive cutaneous anaphylaxis (PCA) is significantlyreduced and can be attributed to a reduction in allergen-IgE inducedmast cell activation and degranulation. In addition, inhibition of deltawith IC 87114 has been shown to significantly ameliorate inflammationand disease in a murine model of asthma using ovalbumin-induced airwayinflammation (Lee et al. FASEB, 20: 455-465 (2006). These data utilizingcompound were corroborated in p110δ^(D910A) mutant mice using the samemodel of allergic airway inflammation by a different group (Nashed etal. Eur. J. Immunol. 37:416-424 (2007)).

There exists a need for further characterization of PI3Kδ function ininflammatory and auto-immune settings. Furthermore, our understanding ofPI3Kδ requires further elaboration of the structural interactions ofp110δ, both with its regulatory subunit and with other proteins in thecell. There also remains a need for more potent and selective orspecific inhibitors of PI3K delta, in order to avoid potentialtoxicology associated with activity on isozymes p110 alpha (insulinsignaling) and beta (platelet activation). In particular, selective orspecific inhibitors of PI3Kδ are desirable for exploring the role ofthis isozyme further and for development of superior pharmaceuticals tomodulate the activity of the isozyme.

SUMMARY

The present invention comprises a new compounds having improvedproperties and biological activity in human PI3Kδ

DETAILED DESCRIPTION

One aspect of the invention relates to compounds having the structures

or any pharmaceutically-acceptable salt thereof.

Another aspect of the invention relates to a method of treatingPI3K-mediated conditions or disorders.

In certain embodiments, the PI3K-mediated condition or disorder isselected from rheumatoid arthritis, ankylosing spondylitis,osteoarthritis, psoriatic arthritis, psoriasis, inflammatory diseases,and autoimmune diseases. In other embodiments, the PI3K-mediatedcondition or disorder is selected from cardiovascular diseases,atherosclerosis, hypertension, deep venous thrombosis, stroke,myocardial infarction, unstable angina, thromboembolism, pulmonaryembolism, thrombolytic diseases, acute arterial ischemia, peripheralthrombotic occlusions, and coronary artery disease. In still otherembodiments, the PI3K-mediated condition or disorder is selected fromcancer, colon cancer, glioblastoma, endometrial carcinoma,hepatocellular cancer, lung cancer, melanoma, renal cell carcinoma,thyroid carcinoma, cell lymphoma, lymphoproliferative disorders, smallcell lung cancer, squamous cell lung carcinoma, glioma, breast cancer,prostate cancer, ovarian cancer, cervical cancer, and leukemia. In yetanother embodiment, the PI3K-mediated condition or disorder is selectedfrom type II diabetes. In still other embodiments, the PI3K-mediatedcondition or disorder is selected from respiratory diseases, bronchitis,asthma, and chronic obstructive pulmonary disease. In certainembodiments, the subject is a human.

Another aspect of the invention relates to the treatment of rheumatoidarthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis,psoriasis, inflammatory diseases or autoimmune diseases comprising thestep of administering a compound according to any of the aboveembodiments.

Another aspect of the invention relates to the treatment of rheumatoidarthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis,psoriasis, inflammatory diseases and autoimmune diseases, inflammatorybowel disorders, inflammatory eye disorders, inflammatory or unstablebladder disorders, skin complaints with inflammatory components, chronicinflammatory conditions, autoimmune diseases, systemic lupuserythematosis (SLE), myestenia gravis, rheumatoid arthritis, acutedisseminated encephalomyelitis, idiopathic thrombocytopenic purpura,multiples sclerosis, Sjoegren's syndrome and autoimmune hemolyticanemia, allergic conditions and hypersensitivity, comprising the step ofadministering a compound according to any of the above or belowembodiments.

Another aspect of the invention relates to the treatment of cancers thatare mediated, dependent on or associated with p110δ activity, comprisingthe step of administering a compound according to any of the above orbelow embodiments.

Another aspect of the invention relates to the treatment of cancers areselected from acute myeloid leukaemia, myelo-dysplastic syndrome,myelo-proliferative diseases, chronic myeloid leukaemia, T-cell acutelymphoblastic leukaemia, B-cell acute lymphoblastic leukaemia,non-hodgkins lymphoma, B-cell lymphoma, solid tumors and breast cancer,comprising the step of administering a compound according to any of theabove or below embodiments.

Another aspect of the invention relates to a pharmaceutical compositioncomprising a compound according to any of the above embodiments and apharmaceutically-acceptable diluent or carrier.

Another aspect of the invention relates to the use of a compoundaccording to any of the above embodiments as a medicament.

Another aspect of the invention relates to the use of a compoundaccording to any of the above embodiments in the manufacture of amedicament for the treatment of rheumatoid arthritis, ankylosingspondylitis, osteoarthritis, psoriatic arthritis, psoriasis,inflammatory diseases, and autoimmune diseases.

The compounds of this invention may have in general several asymmetriccenters and are typically depicted in the form of racemic mixtures. Thisinvention is intended to encompass racemic mixtures, partially racemicmixtures and separate enantiomers and diasteromers.

“Pharmaceutically-acceptable salt” means a salt prepared by conventionalmeans, and are well known by those skilled in the art. The“pharmacologically acceptable salts” include basic salts of inorganicand organic acids, including but not limited to hydrochloric acid,hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid,ethanesulfonic acid, malic acid, acetic acid, oxalic acid, tartaricacid, citric acid, lactic acid, fumaric acid, succinic acid, maleicacid, salicylic acid, benzoic acid, phenylacetic acid, mandelic acid andthe like. When compounds of the invention include an acidic functionsuch as a carboxy group, then suitable pharmaceutically acceptablecation pairs for the carboxy group are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium, quaternaryammonium cations and the like. For additional examples of“pharmacologically acceptable salts,” see infra and Berge et al., J.Pharm. Sci. 66:1 (1977).“Leaving group” generally refers to groups readily displaceable by anucleophile, such as an amine, a thiol or an alcohol nucleophile. Suchleaving groups are well known in the art. Examples of such leavinggroups include, but are not limited to, N-hydroxysuccinimide,N-hydroxybenzotriazole, halides, triflates, tosylates and the like.Preferred leaving groups are indicated herein where appropriate.“Protecting group” generally refers to groups well known in the artwhich are used to prevent selected reactive groups, such as carboxy,amino, hydroxy, mercapto and the like, from undergoing undesiredreactions, such as nucleophilic, electrophilic, oxidation, reduction andthe like. Preferred protecting groups are indicated herein whereappropriate. Examples of amino protecting groups include, but are notlimited to, aralkyl, substituted aralkyl, cycloalkenylalkyl andsubstituted cycloalkenyl alkyl, allyl, substituted allyl, acyl,alkoxycarbonyl, aralkoxycarbonyl, silyl and the like. Examples ofaralkyl include, but are not limited to, benzyl, ortho-methylbenzyl,trityl and benzhydryl, which can be optionally substituted with halogen,alkyl, alkoxy, hydroxy, nitro, acylamino, acyl and the like, and salts,such as phosphonium and ammonium salts. Examples of aryl groups includephenyl, naphthyl, indanyl, anthracenyl, 9-(9-phenylfluorenyl),phenanthrenyl, durenyl and the like. Examples of cycloalkenylalkyl orsubstituted cycloalkylenylalkyl radicals, preferably have 6-10 carbonatoms, include, but are not limited to, cyclohexenyl methyl and thelike. Suitable acyl, alkoxycarbonyl and aralkoxycarbonyl groups includebenzyloxycarbonyl, t-butoxycarbonyl, iso-butoxycarbonyl, benzoyl,substituted benzoyl, butyryl, acetyl, trifluoroacetyl, trichloro acetyl,phthaloyl and the like. A mixture of protecting groups can be used toprotect the same amino group, such as a primary amino group can beprotected by both an aralkyl group and an aralkoxycarbonyl group. Aminoprotecting groups can also form a heterocyclic ring with the nitrogen towhich they are attached, for example, 1,2-bis(methylene)benzene,phthalimidyl, succinimidyl, maleimidyl and the like and where theseheterocyclic groups can further include adjoining aryl and cycloalkylrings. In addition, the heterocyclic groups can be mono-, di- ortri-substituted, such as nitrophthalimidyl. Amino groups may also beprotected against undesired reactions, such as oxidation, through theformation of an addition salt, such as hydrochloride, toluenesulfonicacid, trifluoroacetic acid and the like. Many of the amino protectinggroups are also suitable for protecting carboxy, hydroxy and mercaptogroups. For example, aralkyl groups. Alkyl groups are also suitablegroups for protecting hydroxy and mercapto groups, such as tert-butyl.Silyl protecting groups are silicon atoms optionally substituted by oneor more alkyl, aryl and aralkyl groups. Suitable silyl protecting groupsinclude, but are not limited to, trimethylsilyl, triethylsilyl,triisopropylsilyl, tert-butyldimethylsilyl, dimethylphenylsilyl,1,2-bis(dimethylsilyl)benzene, 1,2-bis(dimethylsilyl)ethane anddiphenylmethylsilyl. Silylation of an amino groups provide mono- ordi-silylamino groups. Silylation of aminoalcohol compounds can lead to aN,N,O-trisilyl derivative. Removal of the silyl function from a silylether function is readily accomplished by treatment with, for example, ametal hydroxide or ammonium fluoride reagent, either as a discretereaction step or in situ during a reaction with the alcohol group.Suitable silylating agents are, for example, trimethylsilyl chloride,tert-butyl-dimethylsilyl chloride, phenyldimethylsilyl chloride,diphenylmethyl silyl chloride or their combination products withimidazole or DMF. Methods for silylation of amines and removal of silylprotecting groups are well known to those skilled in the art. Methods ofpreparation of these amine derivatives from corresponding amino acids,amino acid amides or amino acid esters are also well known to thoseskilled in the art of organic chemistry including amino acid/amino acidester or aminoalcohol chemistry.Protecting groups are removed under conditions which will not affect theremaining portion of the molecule. These methods are well known in theart and include acid hydrolysis, hydrogenolysis and the like. Apreferred method involves removal of a protecting group, such as removalof a benzyloxycarbonyl group by hydrogenolysis utilizing palladium oncarbon in a suitable solvent system such as an alcohol, acetic acid, andthe like or mixtures thereof. A t-butoxycarbonyl protecting group can beremoved utilizing an inorganic or organic acid, such as HCl ortrifluoroacetic acid, in a suitable solvent system, such as dioxane ormethylene chloride. The resulting amino salt can readily be neutralizedto yield the free amine. Carboxy protecting group, such as methyl,ethyl, benzyl, tert-butyl, 4-methoxyphenylmethyl and the like, can beremoved under hydrolysis and hydrogenolysis conditions well known tothose skilled in the art.It should be noted that compounds of the invention may contain groupsthat may exist in tautomeric forms, such as cyclic and acyclic amidineand guanidine groups, heteroatom substituted heteroaryl groups (Y′═O, S,NR), and the like, which are illustrated in the following examples:

and though one form is named, described, displayed and/or claimedherein, all the tautomeric forms are intended to be inherently includedin such name, description, display and/or claim.Prodrugs of the compounds of this invention are also contemplated bythis invention. A prodrug is an active or inactive compound that ismodified chemically through in vivo physiological action, such ashydrolysis, metabolism and the like, into a compound of this inventionfollowing administration of the prodrug to a patient. The suitabilityand techniques involved in making and using prodrugs are well known bythose skilled in the art. For a general discussion of prodrugs involvingesters see Svensson and Tunek Drug Metabolism Reviews 165 (1988) andBundgaard Design of Prodrugs, Elsevier (1985). Examples of a maskedcarboxylate anion include a variety of esters, such as alkyl (forexample, methyl, ethyl), cycloalkyl (for example, cyclohexyl), aralkyl(for example, benzyl, p-methoxybenzyl), and alkylcarbonyloxyalkyl (forexample, pivaloyloxymethyl). Amines have been masked asarylcarbonyloxymethyl substituted derivatives which are cleaved byesterases in vivo releasing the free drug and formaldehyde (Bungaard J.Med. Chem. 2503 (1989)). Also, drugs containing an acidic NH group, suchas imidazole, imide, indole and the like, have been masked withN-acyloxymethyl groups (Bundgaard Design of Prodrugs, Elsevier (1985)).Hydroxy groups have been masked as esters and ethers. EP 039,051 (Sloanand Little, Apr. 11, 1981) discloses Mannich-base hydroxamic acidprodrugs, their preparation and use.

The specification and claims contain listing of species using thelanguage “selected from . . . and . . . ” and “is . . . or . . . ”(sometimes referred to as Markush groups). When this language is used inthis application, unless otherwise stated it is meant to include thegroup as a whole, or any single members thereof, or any subgroupsthereof. The use of this language is merely for shorthand purposes andis not meant in any way to limit the removal of individual elements orsubgroups as needed.

EXPERIMENTAL

The following abbreviations are used:

-   aq.—aqueous-   BINAP—2,2′-bis(diphenylphosphino)-1,1′-binaphthyl-   cond—concentrated-   DCM dichloromethane-   DMF—N,N-dimethylformamide-   Et₂O—diethyl ether-   EtOAc—ethyl acetate-   EtOH—ethyl alcohol-   h—hour(s)-   min—minutes-   MeOH—methyl alcohol-   rt room temperature-   satd—saturated-   THF—tetrahydrofuran

General

Reagents and solvents used below can be obtained from commercialsources. ¹H-NMR spectra were recorded on a Bruker 400 MHz and 500 MHzNMR spectrometer. Significant peaks are tabulated in the order:multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m,multiplet; br s, broad singlet), coupling constant(s) in Hertz (Hz) andnumber of protons. Mass spectrometry results are reported as the ratioof mass over charge, followed by the relative abundance of each ion (inparentheses Electrospray ionization (ESI) mass spectrometry analysis wasconducted on a Agilent 1100 series LC/MSD electrospray massspectrometer. All compounds could be analyzed in the positive ESI modeusing acetonitrile:water with 0.1% formic acid as the delivery solvent.Reverse phase analytical HPLC was carried out using a Agilent 1200series on Agilent Eclipse XDB-C18 5 μm column (4.6×150 mm) as thestationary phase and eluting with acetonitrile:H₂O with 0.1% TFA.Reverse phase semi-prep HPLC was carried out using a Agilent 1100 Serieson a Phenomenex Gemini™ 10 μm C18 column (250×21.20 mm) as thestationary phase and eluting with acetonitrile:H₂O with 0.1%. Chiralcompounds are purified using Isopropanol/Hexane gradient, AD column. Theassignment of chirality is based on the biochemical data.

Example 1 Preparation of4-amino-6-(((1S)-1-(8-fluoro-2-(2-(methyl-sulfonyl)phenyl)-3-quinolinyl)ethyl)amino)-5-pyrimidinecarbonitrileN-(2-fluorophenyl)cinnamamide

To a solution of 2-fluoroaniline (25.0 g, 225 mmol) and potassiumcarbonate (47 g, 337 mmol) in water (112 mL) and acetone (45 mL) at 0°C. was added cinnamoyl chloride (37.0 g, 225 mmol, 1 eq) in acetone (45mL) over 2 h. The reaction was stirred for 1 h at 0° C., and quenchedinto 200 mL of ice-water. The white crystalline solid was filtered andwashed with water. The solid was air dried for 2 h, then washed with 400mL of hexanes. The solid was dried under vacuum overnight to affordN-(2-fluorophenyl)cinnamamide (56 g, 103% yield). ¹H NMR (400 MHz,CDCl₃) δ ppm 8.49 (br t, J=7.8 Hz, 1H), 7.80 (d, J=15.3 Hz, 1H), 7.57(m, 3H), 7.41 (m, 3H), 7.17 (m, 3H), 6.61 (d, J=15.6 Hz, 1H). MassSpectrum (ESI) m/e=242.1 (M+1).

8-fluoroquinolin-2(1H)-one

N-(2-Fluorophenyl)cinnamamide (10.5 g, 44 mmol) was dissolved inchloro-benzene (60 mL) and aluminum trichloride (29.0 g, 218 mmol, 5 eq)was added. The reaction was heated to 125° C. for 3 h and then cooled tort over 45 min. The reaction was poured onto 300 g of ice with stirring,producing a tan solid. The solid was filtered and washed with 100 mL ofwater and 3×100 mL of hexanes and dried under high vacuum. The solid wasextracted with 1 L of DCM and filtered to remove insoluble byproducts.The solvent was removed in vacuo to afford 8-fluoroquinolin-2(1H)-one.¹H NMR (400 MHz, CDCl₃) δ ppm 10.95 (br s, 1H), 7.77 (dd, J=9.8, 1.6 Hz,1H), 7.35 (d, J=7.8 Hz, 1H), 7.27 (ddd, J=10.2, 7.8, 1.2 Hz, 1H), 7.14(td, J=8.0, 5.1 Hz, 1H), 6.76 (d, J=9.4 Hz, 1H).

2-chloro-8-fluoroquinoline

8-Fluoroquinolin-2(1H)-one (26.0 g, 159 mmol) was slurried withphosphoryl trichloride (163 ml, 1.73 mol, 11 eq) and heated to 125° C.for 2 h. The reaction was cooled to rt and poured onto 1.2 L of icewater with vigorous stirring. When the mixture had cooled to rt, theorange solid was filtered and washed with water and dried under vacuumovernight to afford 27 g of crude material. The crude material wasrecrystallized from hexanes by dissolving in ˜700 mL of hexanes atreflux and decanting away from residual tar. The hexane solution wascooled to 0° C. and the precipitate 2-chloro-8-fluoroquinoline wasfiltered. The mother liquor was concentrated in vacuo and recrystallizedfrom hexanes to obtain a second crop of 2-chloro-8-fluoroquinoline (21.3g, 74% total yield).

¹H NMR (400 MHz, CDCl₃) δ ppm 8.14 (dd, J=8.6, 1.2 Hz, 1H), 7.62 (br d,1H), 7.52 (td, J=7.8, 4.7 Hz, 1H), 7.45 (m, 2H).

1-(2-chloro-8-fluoroquinolin-3-yl)ethanol

2-Chloro-8-fluoroquinoline (5.00 g, 27.5 mmol) was dissolved in THF (60mL) and cooled to −78° C. To this solution was added freshly preparedand titrated lithium diisopropylamide (1M solution in THF, 30 mL, 30mmol, 1.1 eq) over 5 min. The reaction was allowed to stir at −78° C.for 20 min, after which time acetaldehyde (2.3 mL, 41.3 mmol, 1.5 eq.)was added via syringe over 30 seconds (exothermic). After 30 min at −78°C., the reaction was quenched with 50% saturated NH₄Cl solution anddiluted with EtOAc. The layers were separated and washed with brine,dried over MgSO₄, and filtered. The crude reaction mixture was depositedon 30 g of silica gel and passed through a plug of 60 g of silica gel,eluted with 8:2 hexanes:EtOAc. Fractions containing product and aclosely (second) eluting regioisomer were collected. The fractions wereconcentrated and the crude solid was slurried in 140 mL of 9:1hexanes:EtOAc at reflux for 30 min. After cooling to rt, the solid wasfiltered and washed with a small amount of cold 9:1 hexanes:EtOAc toafford pure 1-(2-chloro-8-fluoroquinolin-3-yl)ethanol (3.1 g, 13.7 mmol,50% yield). ¹H NMR (400 MHz, CDCl₃) δ ppm 8.43 (br s, 1H), 7.64 (td,J=7.8, 5.1 Hz, 1H), 7.41 (ddd, J=10.2, 7.4, 1.2 Hz, 1H), 5.39 (qdd,J=6.3, 3.9, 0.8 Hz, 1H), 2.22 (d, J=3.9 Hz, 1H), 1.62 (d, J=6.3 Hz, 3H).

1-(2-chloro-8-fluoroquinolin-3-yl)ethanone

To a round-bottomed flask containing toluene (183 mL) was added1-(2-chloro-8-fluoroquinolin-3-yl)ethanol (6.20 g, 27.5 mmol) andmanganese dioxide (19.1 g, 220 mmol, 8 eq). The reaction was heated toreflux for 2 h, cooled to rt, filtered and concentrated. The product wasdiluted with hexanes and filtered to give as a white solid1-(2-chloro-8-fluoroquinolin-3-yl)ethanone (4.43 g, 72% yield). ¹H NMR(400 MHz, CDCl₃) δ ppm 8.40 (d, J=1.6 Hz, 1H), 7.71 (br d, J=8.2 Hz,1H), 7.56 (td, J=7.8, 5.1 Hz, 1H), 7.54 (ddd, J=9.8, 7.8, 1.6 Hz, 1H).Mass Spectrum (ESI) m/e=223.9 (M+1).

(R)-1-(2-chloro-8-fluoroquinolin-3-yl)ethanol

In a round bottomed flask was dissolved (+)-dip-chloride(tm) (17.5 g,540 mmol, 2.2 eq) in anhydrous THF (200 mL) and the solution was cooledto −55° C. (using a dry ice/MeCN bath). To this solution was added1-(2-chloro-8-fluoroquinolin-3-yl)ethanone (5.50 g, 24.5 mmol) as asolution in THF (50 mL). The reaction was allowed to warm to rt slowlyovernight. After this time the reaction was quenched with 10 mL acetoneand 100 mL of 10% Na₂CO₃ and allowed to stir for 2 h at rt. Ethylacetate (750 mL) was added and the layers were separated. The organicphase was washed 3× with a 50% saturated sodium bicarbonate solution andonce with brine. The organic layer was dried over MgSO₄, filtered, andconcentrated. The crude material was concentrated under high vacuum at75° C. to remove pinene. The residue was slurried in 150 mL of hexanesand 150 mL of water for 3 h at rt. A white precipitate formed and wasfiltered and dried to afford 4.9 g of 98% ee product. The solid wasdissolved in 25 mL of boiling EtOAc and 25 mL of hot hexanes was addedto form a precipitate at reflux. The mixture was cooled to −15° C.,filtered, and washed with cold 9:1 hexanes:EtOAc to afford(R)-1-(2-chloro-8-fluoroquinolin-3-yl)ethanol (4.07 g, 73% yield).Chiral HPLC (10% IPA in hexanes, chiralcel AD-H shows product tobe >99.9% ee. Desired enantiomer elutes at 9.6 min, undesired enantiomerelutes at 8.1 min. ¹H NMR (400 MHz, CDCl₃) δ ppm 8.43 (br s), 7.64 (brd, J=8.2 Hz, 1H), 7.50 (td, J=7.8, 4.7 Hz, 1H), 7.41 (ddd, J=10.2, 7.8,1.2 Hz, 1H), 5.40 (qd, J=5.9, 0.8 Hz, 1H), 2.22 (br s, 1H), 1.62 (d,J=6.3 Hz, 3H). Mass Spectrum (ESI) m/e=226.0 (M+1).

(S)-2-(1-(2-chloro-8-fluoroquinolin-3-yl)ethyl)isoindoline-1,3-dione

In a round bottomed flask was combined phthalimide (6.38 g, 43.3 mmol),triphenylphosphine (1.14 g, 43.3 mmol), and(R)-1-(2-chloro-8-fluoroquinolin-3-yl)ethanol (8.15 g, 36.2 mmol) in THF(240 mL). The solution was cooled to 0° C. anddiisopropylazodicarboxylate (DIAD, 8.5 mL, 43.3 mmol) was addeddropwise. The reaction was allowed to warm to rt overnight. The reactionwas concentrated to a volume of ˜100 mL and diluted with 1 L of Et₂O and200 mL of water. The layers were separated and the aqueous layer wasback extracted with 200 mL of Et₂O. The combined organic layers werewashed with 160 mL of brine, dried over MgSO₄, filtered, andconcentrated. Column chromatography using 100% DCM afforded(S)-2-(1-(2-chloro-8-fluoroquinolin-3-yl)ethyl)-isoindoline-1,3-dione(10.5 g, 82% yield). ¹H NMR (400 MHz, CDCl₃) δ ppm 8.60 (br s, 1H), 7.74(m, 2H), 7.64 (m, 3H), 7.45 (td, J=7.8, 4.9 Hz, 1H), 7.35 (ddd, J=10.2,7.8, 1.2 Hz, 1H), 5.89 (q, J=7.2 Hz, 1H), 1.90 (d, J=7.0 Hz, 3H).

2-((1S)-1-(8-fluoro-2-(2-(methylthio)phenyl)quinolin-3-yl)ethyl)isoindoline-1,3-dione

(S)-2-(1-(2-Chloro-8-fluoroquinolin-3-yl)ethyl)isoindoline-1,3-dione(14.0 g, 39.5 mmol), 2-(methylthio)phenylboronic acid (9.95 g, 59.2mmol), and potassium carbonate (16.4 g, 118 mmol) were combined in 300mL of anhydrous DMF under an atmosphere of N₂. The solution was spargedwith N₂ for ˜5 min before adding PdCl₂(dppf)CH₂Cl₂ (3.22 g, 3.95 mmol).The solution was heated at 100° C. for 3 h, and then cooled to 50° C.The solution was concentrated under vacuum to give a brownish residue,which was diluted with EtOAc (600 mL). The organic layers were thenwashed with H₂O (3×80 mL), followed by brine (1×100 mL). The combinedaq. layers were extracted with DCM (3×200 mL). The combined organiclayers were dried over MgSO₄ and then concentrated under vacuum. Theresidue obtained was purified by silica gel flash chromatography elutingwith a gradient of 20% hexane to 40% EtOAc/hexane. The fractionscontaining the pure product were combined and concentrated under vacuumto give2-((1S)-1-(8-fluoro-2-(2-(methylthio)phenyl)quinolin-3-yl)ethyl)isoindoline-1,3-dione(14.6 g, 33.0 mmol, 84% yield) as a light yellow foam. The proton NMRreflects a 53/47 ratio of atropisomers at 25° C. ¹H NMR (500 MHz, CDCl₃)δ ppm 8.71 (br s, 0.53H), 8.65 (br s, 0.47H), 7.79 (m, 1H), 7.66 (s,4H), 7.55 (m, 1H), 7.45-7.27 (series of m, 3.6H), 6.87 (m, 1.4H), 5.70(q J=6.4 Hz, 0.47H), 5.63 (q, J=6.8 Hz, 0.53H), 2.47 (br s, 1.4H), 1.91(m, 3H), 1.52 (br s, 1.6H). Mass Spectrum (ESI) m/e=443.2 (M+1).

2-((1S)-1-(8-fluoro-2-(2-(methylsulfonyl)phenyl)-3-quinolinyl)ethyl)-1H-isoindole-1,3(2H)-dione

To 70 mL of DCM was added 13.56 g (1.2 g/1 mmol of substrate) of wetMontmorillonite (˜0.2 g H₂O/1 g of clay), and oxone (17.37 g, 28.2mmol)¹. To this suspension was added a solution of2-((1S)-1-(8-fluoro-2-(2-(methylthio)-phenyl)quinolin-3-yl)ethyl)isoindoline-1,3-dione(5.0 g, 11.3 mmol) dissolved in DCM (10 mL). The slurry was stirred atrt for 72 h and then filtered through a fritted funnel. The solids werewashed with DCM (−600 mL) and the filtrates were concentrated undervacuum to give2-((1S)-1-(8-fluoro-2-(2-(methylsulfonyl)phenyl)-3-quinolinyl)ethyl)-1H-isoindole-1,3(2H)-dione7.11 g (97%) as a light yellow solid. The proton NMR reflects a 59/41ratio of atropisomers at 25° C. ¹H-NMR (500 MHz, CDCl₃) δ ppm 8.86 (s,0.59H), 8.79 (s, 0.41H), 8.26 (d, J=7.8 Hz, 0.41H), 8.04 (d, J=7.8 Hz,0.59H), 7.89-7.36 (series of m, 9.6H), 7.20 (d, J=7.6 Hz, 0.41H), 5.70(q, J=7.1 Hz, 0.59H), 5.54 (q, J=7.3 Hz, 0.41H), 3.19 (s, 1.75H), 3.15(s, 1.25H), 1.98 (d, J=7.3 Hz, 1.75H), 1.85 (d, J=7.1 Hz, 1.25H). MassSpectrum (ESI) m/e=475.0 (M+1).

-   1. Hirano, M.; Tomaru, J.; Morimoto, T. Bull. Chem. Soc. Jpn., 1991,    64, 3752-54.

(1S)-1-(8-fluoro-2-(2-(methylsulfonyl)phenyl)-3-quinolinyl)ethanamine

2-((1S)-1-(8-Fluoro-2-(2-(methylsulfonyl)phenyl)quinolin-3-yl)ethyl)isoindoline-1,3-dione(9.10 g, 19.2 mmol), and hydrazine hydrate (9.32 mL, 192 mmol) wereadded to EtOH (190 mL). After heating at 65° C. for 3 h, the resultantslurry was cooled to rt, diluted with 900 mL of EtOAc, and filteredthrough a fitted funnel. The filtrate was washed with H₂O (3×200 mL),brine (1×200 mL) and then dried over MgSO₄ before being concentratedunder vacuum to give(1S)-1-(8-fluoro-2-(2-(methylsulfonyl)phenyl)quinolin-3-yl)ethanamine(6.06 g, 17.6 mmol, 92% yield) as a light orange solid. The proton NMRreflects a 73/27 ratio of atropisomers at 25° C. ¹H NMR (500 MHz, CDCl₃)δ ppm 8.55 (d, J=1.5 Hz, 0.73H), 8.50 (d, J=1.5 Hz, 0.27H), 8.26 (dd,J=8.1, 1.2 Hz, 0.27H), 8.23 (dq, J=7.8 1.2 Hz, 0.73H), 7.73 (m, 3H),7.52 (m, 1.27H), 7.40 (m, 1.73H), 4.16 (q, J=6.6 Hz, 0.27H), 4.03 (q,J=6.4 Hz, 0.73H), 3.36 (s, 0.79H), 3.17 (s, 2.21H), 1.36 (m, 3H). MassSpectrum (ESI) m/e=345.2 (M+1).

4-amino-6-(((1S)-1-(8-fluoro-2-(2-(methylsulfonyl)phenyl)-3-quinolinyl)-ethyl)amino)-5-pyrimidinecarbonitrile

(1S)-1-(8-Fluoro-2-(2-(methylsulfonyl)phenyl)quinolin-3-yl)ethanamine(9.16 g, 26.6 mmol), N-ethyl-N-isopropylpropan-2-amine (13.7 mL, 80mmol), and 4-amino-6-chloropyrimidine-5-carbonitrile (4.32 g, 27.9 mmol)were combined in n-butanol (67 mL) and then heated to 110° C. under anatmosphere of N₂. After 3 h at 110° C., the temperature of the reactionwas increased to 120° C. for 2 h. After cooling in an ice bath, themixture was filtered leaving 14 g of a brownish solid. The filtrate washeated to 120° C. for 3 h, cooled to 40° C. and concentrated undervacuum leaving a brown oil. The oil and solids were combined andpurified on a silica gel flash column eluting with 2% MeOH/DCM. Thefractions containing the pure product were combined and concentratedunder vacuum to give a light yellowish solid. The fractions containingthe impure product were combined and purified on a silica gel flashcolumn eluting with a gradient of 1.5% MeOH/DCM to 2% MeOH/DCM. Thefractions containing the pure product were combined and concentratedunder vacuum to give a light yellowish solid. The combined pure solidswere dissolved in EtOH with heating (˜60° C.), and then concentratedunder vacuum, repeated the dissolution into EtOH followed byconcentration under vacuum. The solids obtained were then dried on thevacuum line at 120° C. until residual ethanol was below 0.5% by weight.The solid obtained was4-amino-6-(((1S)-1-(8-fluoro-2-(2-(methylsulfonyl)phenyl)-3-quinolinyl)ethyl)-amino)-5-pyrimidinecarbonitrile,10.3 g (84% yield). The proton NMR reflects a 87/13 ratio ofatropisomers at 25° C. ¹H NMR (500 MHz, DMSO-d6) δ ppm 8.80 (br s,0.13H), 8.58 (br s, 0.87H), 8.13 (dd, J=7.7, 1.2 Hz, 0.87H), 7.95-7.52(series of m, 8H), 7.30-6.99 (br m, 2H), 6.82 (d, J=8.3 Hz, 1H), 5.62(quintet, J=6.9 Hz, 0.13H), 5.22 (quintet, J=6.9 Hz, 0.87H), 3.37 (s,2.57H), 3.31 9s, 0.43H), 1.60 (d, J=6.9 Hz, 0.4H), 1.35 (d, J=6.9 Hz,2.6H). Mass Spectrum (ESI) m/e=463.1 (M+1).

Example 2 Preparation of(S)-4-amino-6-((1-(7-fluoro-2-(2-(methylsulfonyl)-phenyl)quinolin-3-yl)ethyl)amino)pyrimidine-5-carbonitrile2-chloro-7-fluoroquinoline-3-carbaldehyde

POCl₃ (0.837 L, 9.17 mmol) was added dropwise to DMF (253 mL, 3.28 mmol)in a three-necked flask equipped with mechanical stirrer at 0° C. Thesemi-solid mixture was stirred at rt for 30 min andN-(3-fluorophenyl)acetamide (200 g, 1.31 mol) was added in one portion.The resulting mixture was heated at 75° C. over night. After cooling tort, the reaction mixture was poured carefully into ice-water (9 kg). Theresulted solid was filtered, washed with water, NaHCO₃, and dried in theair. The crude mixture (200 g, 73%) was recrystallized in EtOAc (5 L) togive 2-chloro-7-fluoroquinoline-3-carbaldehyde as off white needles (150g). Mass Spectrum (ESI) m/e=210 (M+1).

1-(2-chloro-7-fluoroquinolin-3-yl)ethanol

A suspension of 2-chloro-7-fluoroquinoline-3-carbaldehyde (44.7 g, 213mmol) in THF (600 mL) was treated with MeMgBr (78.0 mL, 234 mmol 1.1 eq)at −20° C. After stirring overnight, the reaction was quenched withNH₄Cl solution and extracted with Et₂O (300 mL and 100 mL). The organiclayers were washed with water, brine, dried over Na₂SO₄, concentratedand recrystallized from EtOAc (100 mL) and hexane (1 L). A pale yellowsolid of 1-(2-chloro-7-fluoroquinolin-3-yl)-ethanol was obtained (41 g,85%). Mass Spectrum (ESI) m/e=226 (M+1).

1-(2-chloro-7-fluoroquinolin-3-yl)ethanone

1-(2-Chloro-7-fluoroquinolin-3-yl)ethanol (7.7 g, 34 mmol), MnO₂ (30 g,10 eq) and Toluene (200 mL) were heated to reflux for 2 h. LC-MS showedcompletion of the reaction. Filtration followed with removal of solventgave an off-white solid of 1-(2-chloro-7-fluoroquinolin-3-yl)ethanone(6.2 g, 81%). Mass Spectrum (ESI) m/e=224 (M+1).

(R)-1-(2-chloro-7-fluoroquinolin-3-yl)ethanol

A solution of 1-(2-chloro-7-fluoroquinolin-3-yl)ethanone (164 g, 733mmol) in THF (1.34 L) was added to a solution of (+)-DIP-Cl (517.5 g,2.2 eq) in THF (3.5 L) at −45° C. (dry ice and acetonitrile) dropwise.The reaction was slowly warmed up to rt overnight. The reaction was thenquenched with acetone (750 mL) and 10% Na₂CO₃ (750 mL) at 0° C. andstirred at rt for 1 h before the addition of EtOAc (3.5 L). The mixturewas warmed up to rt and washed with 10% Na₂CO₃ and water. The organiclayer was dried with brine, concentrated and treated with hexane (1.0 L)and water (1.8 L). The mixture was stirred at rt for 40 min andfiltered. The white solid was washed with water and hexane, dried in theair over night (143 g). This material was dried on the roto-evaporatorat 60° C. for 4 h under high vacuum (2 mm Hg) to give a white powder of(R)-1-(2-chloro-7-fluoroquinolin-3-yl)ethanol (122 g, 73.7%). ChiralHPLC on AD column (isopropanol in hexane, 10%) showed ee >99%. MassSpectrum (ESI) m/e=226 (M+1).

S)-2-(1-(2-chloro-7-fluoroquinolin-3-yl)ethyl)isoindoline-1,3-dione

To a mixture of (R)-1-(2-chloro-7-fluoroquinolin-3-yl)ethanol (3.03 g,13.4 mmol), phthalimide (2.37 g, 1.20 eq) and PPh₃ (4.23 g, 1.20 eq) inTHF (70 mL) was added DIAD (3.13 mL, 1.20 eq) dropwise at 0° C. Themixture was then stirred at rt overnight before partitioning betweenEtOAc (200 mL) and water (200 mL). The organic layer was separated,washed with water, brine, dried over Na₂SO₄ and concentrated. Theresidue was purified by column chromatography on silica gel (DCM) togive a white foam of(S)-2-(1-(2-chloro-7-fluoroquinolin-3-yl)ethyl)isoindoline-1,3-dione(3.8 g, 80%). Mass Spectrum (ESI) m/e=355 (M+1).

(S)-tert-butyl (1-(2-chloro-7-fluoroquinolin-3-yl)ethyl)carbamate

To a solution of(S)-2-(1-(2-chloro-7-fluoroquinolin-3-yl)ethyl)isoindoline-1,3-dione(1.0 g, 2.8 mmol) in EtOH (10 mL) was added dropwise NH₂NH₂ (10 eq, 0.88mL) at rt before warmed up to 90° C. for 30 min. After cooling to rt,the reaction mixture was concentrated, partitioned between EtOAc (20 mL)and water (5 mL). The organic layer was separated, washed with water,brine, dried over Na₂SO₄ and concentrated to give a colorless oil, whichwas dissolved in THF (10 mL) and treated with BOC₂O (1.1 eq, 0.68 g) andTEA (1.0 eq, 0.39 mL) at reflux. After cooling to rt, the reactionmixture was concentrated and purified by column chromatography on silicagel (EtOAc/hexane, 1/9) to give a white solid (S)-tert-butyl(1-(2-chloro-7-fluoroquinolin-3-yl)ethyl)carbamate (0.70 g, 76%). MassSpectrum (ESI) m/e=325 (M+1).

(S)-tert-butyl(1-(7-fluoro-2-(2-(methylthio)phenyl)quinolin-3-yl)ethyl)-carbamate

A mixture of (S)-tert-butyl(1-(2-chloro-7-fluoroquinolin-3-yl)ethyl)carbamate (382 mg, 1.2 mmol),2-(methylthio)phenylboronic acid (257 mg, 1.3 eq), Na₂CO₃ (623 mg, 5.0eq), Pd(PPh₃)₄ (93 mg, 5%), MeCN (9 mL) and water (3 mL) was heated to85° C. under N₂ overnight. After cooling to rt, the reaction waspartitioned between EtOAc (10 mL) and water (5 mL). The organic layerwas separated, washed, dried and concentrated. The residue was purifiedby column chromatography on silica gel to give a white solid(S)-tert-butyl(1-(7-fluoro-2-(2-(methylthio)phenyl)quinolin-3-yl)ethyl)carbamate (460mg, 94.8%). Mass Spectrum (ESI) m/e=413 (M+1).

(S)-tert-butyl(1-(7-fluoro-2-(2-(methylsulfonyl)phenyl)quinolin-3-yl)ethyl)-carbamate

Under an N₂ atmosphere, (S)-tert-butyl(1-(7-fluoro-2-(2-(methylthio)phenyl)-quinolin-3-yl)ethyl)carbamate (412mg, 999 μmol) was dissolved in acetone (3.00 ml, 40.8 mmol) and water (3mL), and NMO (351 mg, 3.00 mmol) was added followed by OsO₄ (12.7 mg,0.05 mmol). The reaction was allowed to stir at rt overnight. LC-MSshowed the reaction not to be complete. The reaction was treated withOsO₄ (12.7 mg, 0.05 mmol) again and stirred overnight. LC-MS showed onlya trace of the starting reagent. The reaction was quenched by theaddition of 5 mL of a saturated sodium thiosulfate solution. Thereaction was diluted with EtOAc and washed with 5% Na₂CO₃. The organiclayer was then washed with brine, dried (Na₂SO₄), filtered andconcentrated in vacuo. The crude residue was purified eluting with 30%to 60% EtOAc in hexanes to provide a white solid (S)-tert-butyl(1-(7-fluoro-2-(2-(methylsulfonyl)phenyl)quinolin-3-yl)-ethyl)carbamate(440 mg, 99%). Mass Spectrum (ESI) m/e=445 (M+1).

(S)-4-amino-6-((1-(7-fluoro-2-(2-(methylsulfonyl)phenyl)quinolin-3-yl)ethyl)-amino)pyrimidine-5-carbonitrile

To (S)-tert-butyl(1-(7-fluoro-2-(2-(methylsulfonyl)phenyl)quinolin-3-yl)ethyl)-carbamate(440 mg, 1.0 mmol) was added HCl, 4M (1 mL). The resulting mixture wasstirred at rt for 1 h. Solvent was removed and the crude(S)-1-(7-fluoro-2-(2-(methylsulfonyl)phenyl)quinolin-3-yl)ethanamine wasused without further workup. A solution of(1S)-1-(7-fluoro-2-(2-(methylsulfonyl)phenyl)-quinolin-3-yl)ethanamine(100 mg, 290 μmol), 4-amino-6-chloropyrimidine-5-carbonitrile (45 mg,290 μmol) and DIEA (101 μl, 581 μmol) in DMF (4 mL) was heated to 100°C. overnight. Solvent was removed under reduce pressure and purified viapreparatory. TLC using 3% of MeOH/DCM to afford a white powder of(S)-4-amino-6-((1-(7-fluoro-2-(2-(methylsulfonyl)phenyl)quinolin-3-yl)ethyl)-amino)pyrimidine-5-carbonitrile(6.9 mg, 5.1%). ¹H NMR (500 MHz, CD₃OD) δ ppm 1.51 (d, J=7.09 Hz, 3H)3.17 (s, 3H) 5.43 (d, J=6.85 Hz, 1H) 7.48 (d, J=2.45 Hz, 1H) 7.60-7.63(m, 1H) 7.66-7.69 (m, 2H) 7.73 (d, J=1.47 Hz, 1H) 7.81 (s, 1H) 8.07 (dd,J=9.05, 6.11 Hz, 1H) 8.13-8.17 (m, 1H) 8.49 (s, 1H). Mass Spectrum (ESI)m/e=463 (M+1).

Example 3 Preparation of4-amino-6-((S)-1-(7-fluoro-2-(3-fluorophenyl)-quinolin-3-yl)ethylamino)pyrimidine-5-carbonitrile2-((S)-1-(7-fluoro-2-(3-fluorophenyl)quinolin-3-yl)ethyl)isoindoline-1,3-dione

To a solution of(S)-2-(1-(2-chloro-7-fluoroquinolin-3-yl)ethyl)isoindoline-1,3-dione(150 mg, 423 μmol), 3-fluorophenylboronic acid (65 mg, 465μmol) andsodium carbonate (90 mg, 846μmol) in MeCN (8 mL) and water (2 mL) waspurged with N₂ followed by the addition of Pd(PPh₃)₄ (24 mg, 21 μmol)and the resulting mixture was stirred at 90° C. overnight. The reactionmixture was diluted with EtOAc washed with water, brine and dried overNa₂SO₄. Purification using preparatory TLC eluted with 100% EtOAcafforded2-4(S)-1-(7-fluoro-2-(3-fluorophenyl)quinolin-3-yl)ethyl)carbamoyl)benzoicacid (120 mg, 66%). Mass Spectrum (ESI) m/e=433 (M+1).24(S)-1-(7-Fluoro-2-(3-fluorophenyl)-quinolin-3-yl)ethyl)carbamoyl)benzoicacid was dissolved in EtOH (2 mL) and cond HCl (0.1 mL) was added. Theresulting solution was heated to 80° C. for 4 h. Solvent was removed andsatd NaHCO₃ was added to the reaction mixture and extracted with EtOAc.The organic layer was washed with brine, dried over Na₂SO₄, concentratedand purified via preparatory TLC using 3% MeOH/DCM saturated with NH₃gas to afford a white solid of2-((S)-1-(7-fluoro-2-(3-fluorophenyl)quinolin-3-yl)ethyl)isoindoline-1,3-dione(85 mg, 49%). Mass Spectrum (ESI) m/e=415 (M+1).

4-amino-6-((S)-1-(7-fluoro-2-(3-fluorophenyl)quinolin-3-yl)ethylamino)-pyrimidine-5-carbonitrile

To a solution of2-((S)-1-(7-fluoro-2-(3-fluorophenyl)quinolin-3-yl)ethyl)-isoindoline-1,3-dione(60.0 mg, 145 μmol) in EtOH (2 mL) was added NH₂NH₂ (1.0 mL, 1.45 mmol,10 eq) and the resulting solution was heated to 80° C. for 2 h. Aprecipitate was filtered away and the filtrate was removed under reducepressure to afford(1S)-1-(7-fluoro-2-(3-fluorophenyl)quinolin-3-yl)ethanamine. To thecrude residue of(1S)-1-(7-fluoro-2-(3-fluorophenyl)quinolin-3-yl)-ethanamine in DMF (2mL) was added 4-amino-6-chloropyrimidine-5-carbonitrile (22 mg, 145μmol) and DIEA (0.06 mL, 319 μmol). The resulting mixture was heated to100° C. overnight. Solvent was removed under reduce pressure andpurified via preparatory HPLC using 15-60% MeCN/H₂O w/0.01% TFA toafford a white solid of4-amino-6-((S)-1-(7-fluoro-2-(3-fluorophenyl)-quinolin-3-yl)ethylamino)pyrimidine-5-carbonitrile(9.3 mg, 16%). ¹H NMR (500 MHz, CD₃OD) δ ppm 1.63 (d, J=6.85 Hz, 3H)5.70 (q, J=7.01 Hz, 1H) 7.25-7.34 (m, 1H) 7.44-7.54 (m, 2H) 7.55-7.63(m, 2H) 7.74 (dd, J=9.78, 2.45 Hz, 1H) 8.05 (s, 1H) 8.18 (dd, J=9.05,5.87 Hz, 1H) 8.72 (s, 1H). Mass Spectrum (ESI) m/e=403 (M+1).

Example 4 Preparation of(S)-4-amino-6-((1-(7-fluoro-2-(pyridin-2-yl)-quinolin-3-yl)ethyl)amino)pyrimidine-5-carbonitrile2-((S)-1-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)isoindoline-1,3-dione

A mixture of(S)-2-(1-(2-chloro-7-fluoroquinolin-3-yl)ethyl)isoindoline-1,3-dione(85.6 g, 241 mmol), Pd(PPh₃)₄ (14 g, 12 mmol, 0.05 eq) and2-(tributylstannyl)-pyridine (107 g, 289 mmol, 1.2 eq) in dioxane (3.0L) was heated to 90° C. under N₂. After stirring overnight, LC-MS showed30% completion. The reaction mixture was heated to 101° C. foradditional 2 days. The reaction mixture was then cooled to rt, decantedand the remaining 200 mL solution filtered to remove Pd residue. Thecombined solvents were concentrated to 300 mL and filtered to give a tansolid, which was washed with EtOAc/hexane (1/1) and dried to provide(82.1 g). The mother liquor was concentrated to 100 mL and treated withEtOAc/hexane, 1/1 (200 mL) to give a second crop of product (2.2 g).Overall a tan solid of2-((S)-1-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)isoindoline-1,3-dionewas obtained (84.3 g, 88%). Mass Spectrum (ESI) m/e=398 (M+1).

(S)-1-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethanamine

To a slurried2-((S)-1-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethyl)isoindoline-1,3-dione(2.1 g, 5.3 mmol) in anhydrous ethanol (15 mL) was added NH₂NH₂ (0.85 g,26 mmol) dropwise over 5 minutes. The reaction mixture was heated to 90°C. for 30 min and cooled to rt. The reaction mixture was filtered andwashed with EtOAc. The resulting EtOAc solution was washed with water,brine and dried over Na₂SO₄. Removal of solvents gave a tan oil of(S)-1-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethanamine (1 g, 71%).Mass Spectrum (ESI) m/e=468 (M+1).

4-amino-6-((S)-1-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethylamino)-pyrimidine-5-carbonitrile

A mixture of (S)-1-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethanamine(85 mg, 0.32 mmol), 4,6-dichloro-5-cyanopyrimidine (55 mg, 0.32 mmol,1.0 eq) and N,N-diisopropylethylamine (68 μl, 0.38 mmol, 1.2 eq) in THF(3 mL) was stirred at rt for 30 min before heating to 50° C. After 4 h,the mixture was concentrated and purified by column chromatography(EtOAc/1/1) to give a white solid, which was treated with saturated NH₃in dioxane (3 mL) in sealed tube at 110° C. overnight. The reactionmixture was concentrated and purified by reverse phase HPLC(MeCN/H₂O/0.1% TFA) and lyophilized to give a white powder4-amino-6-((S)-1-(7-fluoro-2-(pyridin-2-yl)quinolin-3-yl)ethylamino)pyrimidine-5-carbonitrile(19 mg, 15%). ¹H NMR (500 MHz, CD₃OD) δ ppm 1.78 (d, J=6.85 Hz, 3H) 5.82(q, J=6.85 Hz, 1H) 7.71 (td, J=8.80, 2.45 Hz, 1H) 7.89 (dd, J=9.66, 2.32Hz, 1H) 8.10 (ddd, J=7.83, 5.62, 0.98 Hz, 1H) 8.19 (s, 1H) 8.27 (dd,J=9.05, 5.87 Hz, 1H) 8.45 (d, J=7.83 Hz, 1H) 8.63 (td, J=7.95, 1.47 Hz,1H) 8.94 (s, 1H) 9.03-9.09 (m, 1H). Mass Spectrum (ESI) m/e=386 (M+1).

Example 5 Preparation of(S)-4-amino-6-((1-(5-chloro-3-(2-(methylsulfonyl)-phenyl)quinoxalin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile2-((3-chloro-2-nitrophenyl)amino)butanoic acid

A mixture of 1-chloro-3-fluoro-2-nitrobenzene (2.00 Kg, 11.4 mol),2-aminobutyric acid (1.22 kg, 11.8 mol) and K₂CO₃ (1.58 Kg, 11.4 mol) inanhydrous DMSO (4.2 L) was heated at 80° C. for 16 h (after the reactioninitiated, the internal temperature went up to 110° C.). At this timeLC-MS analysis showed that the reaction was complete. After cooling tort, the reaction mixture was carefully poured into water (10 L) withvigorous stirring. The aq. layer was washed with methyl tert-butyl ether(2×5 L) to remove organic impurities. The aq. layer was then acidifiedto pH ˜1.5 with cond HCl to give an orange solid. The orange solid wascollected by filtration, washed with water (2×4 L) and air-dried to give2-((3-chloro-2-nitrophenyl)amino)butanoic acid (2.85 Kg), which was usedas such in the next step. ¹H NMR (DMSO-d₆) δ ppm 7.35 (t, J=8.3 Hz, 1H),6.82-6.91 (m, 2H), 6.25 (d, J=7.6 Hz, 1H), 4.14 (td, J=7.4, 5.4 Hz, 1H),3.4 (br. s., 1H), 1.74-1.92 (m, 2H), 0.88 (t, J=7.4 Hz, 3H). MassSpectrum (ESI) m/z=259.2 (M+1).

8-chloro-3-ethyl-3,4-dihydroquinoxalin-2(1H)-one

To a solution of 2-((3-chloro-2-nitrophenyl)amino)butanoic acid (1.5 Kg,5.8 mol), 4 N aq. HCl (4.35 L, 17.4 mol) and EtOH (5.3 L) was addedSnCl₂.2H₂O (3.93 Kg, 17.4 mol). The reaction mixture was heated toreflux for 5 h. At this time LC-MS analysis showed that the reaction wascomplete. After cooling to rt the reaction mixture was evaporated underreduced pressure. The resulting residue was cooled to 0° C. using anice-water bath and an aqueous solution of 10 N KOH (12 L, 180 mol) wascarefully added with vigorous stirring. After filtration to removeinsoluble solids, the filtrate was extracted with DCM (2×10 L) washedwith water, brine, dried over anhydrous Na₂SO₄, and evaporated underreduced pressure to give8-chloro-3-ethyl-3,4-dihydroquinoxalin-2(1H)-one (1.1 Kg) as a yellowsolid, which was used without further purification. ¹H NMR (DMSO-d₆) δppm 9.70 (s, 1H), 6.65-6.83 (m, 3H), 6.31-6.44 (m, 1H), 3.66-3.72 (m,1H), 1.53-1.71 (m, 2H), 0.92 (t, J=7.4 Hz, 3H). Mass Spectrum (ESI)m/z=211.2 (M+1).

8-chloro-3-ethylquinoxalin-2(1H)-one

To a solution of 8-chloro-3-ethyl-3,4-dihydroquinoxalin-2(1H)-one (1.30Kg, 6.17 mol) in anhydrous 1,4-dioxane (15 L) was added DDQ (1.47 Kg,6.48 mol). The reaction mixture was stirred for 3 h (the internaltemperature went up to 45° C. after DDQ addition). After this time LC-MSanalysis showed that the reaction was complete. The mixture wasevaporated under reduced pressure to give a brown residue. To thisresidue was added 2M aqueous NaOH to adjust the pH to 7-8. The resultingyellow solid was collected by filtration, suspended in saturated aq.NaHCO₃, stirred for 1 h, and filtered to give a light green solid. Thelight green solid was suspended in saturated aqueous NaHCO₃, stirred,filtered and washed with saturated aqueous NaHCO₃ and water to give anoff-white solid. The off-white solid was suspended in water, mixed well,filtered, washed with water, air-dried overnight, and dried under highvacuum at 50° C. to give 8-chloro-3-ethylquinoxalin-2(1H)-one (1 Kg). ¹HNMR (DMSO-d₆) δ ppm 7.69-7.75 (m, 1H), 7.59-7.65 (m, 1H), 7.29 (t, J=8.0Hz, 1H), 2.84 (q, J=7.4 Hz, 2H), 1.23 (t, J=7.4 Hz, 3H). Mass Spectrum(ESI) m/z=209.1 (M+1).

3,5-dichloro-2-ethylquinoxaline

A thick slurry of 8-chloro-3-ethylquinoxalin-2(1H)-one (1.00 Kg, 4.79mol) in POCl₃ (2.68 L, 28.8 mol) was stirred at 100° C. for 2 h. At thistime LC-MS analysis showed that the reaction was complete. After theremoval of most of the POCl₃ under reduced pressure, the residue waspoured carefully into ice-water and neutralized with a combination of 2Maq. NaOH and saturated aq. NaHCO₃. The resulting suspension wasextracted with DCM (3×4 L). The organic phase was washed with brine,dried over anhydrous Na₂SO₄, filtered, and evaporated under reducedpressure. The crude was purified by flash column chromatography elutingwith hexane/EtOAc (30/1) to give 3,5-dichloro-2-ethylquinoxaline (840 g)as a white solid. ¹H NMR (DMSO-d₆) δ ppm 8.01-8.08 (m, 2H), 7.84 (t,J=8.0 Hz, 1H), 3.10-3.17 (d, J=7.3 Hz, 2H), 1.36 (t, J=7.3 Hz, 3H). MassSpectrum (ESI) m/z=227.1 (M+1).

2-(1-bromoethyl)-3,5-dichloroquinoxaline

3,5-Dichloro-2-ethylquinoxaline (1.10 Kg, 4.84 mol) was dissolved inCCl₄ (4.4 L) at rt. 1,3-dibromo-5,5-dimethylhydantoin (762 g, 2.66 mol)and benzoyl peroxide (116 g, 0.48 mol) were then added. The resultingsuspension was heated at reflux for 2 h. At this time LC-MS analysisshowed that the reaction was complete. After cooling to rt whitecrystals formed in the reaction vessel. The white crystals werecollected by filtration, washed with saturated aqueous NaHCO₃ (3×5 L),and dried under high vacuum to give2-(1-bromoethyl)-3,5-dichloroquinoxaline as a white solid (1.05 Kg). ¹HNMR (DMSO-d₆) δ ppm 8.04-8.12 (m, 2H), 7.78-7.90 (m, 1H), 5.76-5.84 (m,1H), 2.09 (d, J=6.7 Hz, 3H). Mass Spectrum (ESI) m/z=304.8 [(M+1)(⁷⁹Br)], 306.9 [(M+1) (8′Br)].

2-(1-(3,5-dichloroquinoxalin-2-yl)ethyl)isoindoline-1,3-dione

To a solution of 2-(1-bromoethyl)-3,5-dichloroquinoxaline (1.00 Kg, 3.27mol) in DMF (8.2 L) was added potassium phthalimide (1.21 Kg, 6.54 mol).The reaction mixture was stirred for 3 h. At this time LC-MS analysisshowed that the reaction was complete. The mixture was transferred to a50 L separatory funnel and water (12 L) and EtOAc (6 L) were added to itwith stirring. A large amount of white solid crashed out and wascollected by filtration. The aqueous. phase was separated and extractedagain with EtOAc (2×4 L). The combined organic layers were washed withbrine, dried over anhydrous Na₂SO₄, filtered, and the volume was reducedto 5 L under reduced pressure. After cooling to rt, the product wasobtained as a white solid. The white solid was filtered, triturated withhexane and dried under vacuum to give2-(1-(3,5-dichloroquinoxalin-2-yl)ethyl)isoindoline-1,3-dione (1.24 Kg).¹H NMR (DMSO-d₆) δ ppm 8.02-8.21 (2H, m), 7.83-8.02 (m, 5H), 5.89 (q,J=6.8 Hz, 1H), 1.87 (d, J=6.8 Hz, 3H). Mass Spectrum (ESI) m/z=372.0(M+1).

(S)-2-(1-(3,5-dichloroquinoxalin-2-yl)ethyl)isoindoline-1,3-dione

Racemic 2-(1-(3,5-dichloroquinoxalin-2-yl)ethyl)isoindoline-1,3-dione (1Kg) was purified on a Novasep HPLC unit. The operating conditions usedfor the separation process were:

Column: CHIRALPAK® AS 20 μM, 11 cm id×25 cm L

Mobile Phase Hexane-IPA 70-30

Flow rate: 400 mL/min

Temperature: 25° C.

UV detection: 340 nm

Solubility was 1 g/L in the mobile phase. Stirring and heating wererequired to keep the sample in solution. The solution was filteredbefore use. Injection volume was 510 mL every 8.0 minutes. The fractionscollected from the chromatographic process were evaporated using Artisanthin-film evaporators and rotary evaporators at 45° C. After solventremoval, the product was dried to constant weight in vacuum oven at 40°C. to give:(S)-2-(1-(3,5-dichloro-quinoxalin-2-yl)ethyl)isoindoline-1,3-dione,1^(st) enantiomer (425.7 g, 85.1% yield, 98.7% e.e.) and(R)-2-(1-(3,5-dichloroquinoxalin-2-yl)ethyl)isoindoline-1,3-dione,2^(nd) enantiomer (432.5 g, 86.2% yield, 97.7% e.e.).

2-((1S)-1-(5-chloro-3-(2-(methylthio)phenyl)quinoxalin-2-yl)ethyl)isoindoline-1,3-dione

A 5 L three-necked round-bottomed flask equipped with a mechanicalstirrer, a condenser, a nitrogen gas inlet and a temperature probe wascharged with DMF (2.16 L),(S)-2-(1-(3,5-dichloroquinoxalin-2-yl)ethyl)isoindoline-1,3-dione (273g, 733 mmol) and 2-(methylthio)phenylboronic acid (136 g, 807 mmol). Tothe mixture, was added potassium carbonate (203 g, 147 mmol) and[1,1-bis(di-phenylphosphino)ferrocene]palladium(ii) chloride, complexwith DCM (29.9 g, 36.7 mmol). The mixture was vacuum purged with N₂(2×), and heated to 100° C. The reaction was monitored by LC-MS, anddeemed complete after 4 h. The reaction was cooled to rt (21° C.), andthen divided into two batches. Each batch was partitioned between EtOAc(1.08 L) and water (1.35 L) in a 4 L separatory funnel. After phaseseparation, the organic layer was washed with brine (2×500 mL) andconcentrated to afford the crude product. The combined batches of crudematerial were loaded on silica gel and purified by flash chromatography(ISCO/RediSep™) (hexane:EtOAc=10:0 to 6:4) to provide the product as alight brown solid 320 g with 98% LC purity and 95% yield.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.15-8.31 (m, 1H), 8.07 (m, 1H), 7.93(m, 1H), 7.77 (m, 2H), 7.64 (br.s., 2H), 7.16-7.55 (m, 3H), 6.58-7.02(m, 1H), 5.72-5.98 (m, 1H), 3.29 (s, 3H), 1.79 (d, J=6.9 Hz, 3H) MassSpectrum (ESI) m/z=460.0 (M+1).

2-((1S)-1-(5-chloro-3-(2-(methylsulfonyl)phenyl)quinoxalin-2-yl)ethyl)-isoindoline-1,3-dione

A 5 L three-necked round-bottomed flask equipped with an additionfunnel, nitrogen inlet, overhead stirrer and thermocouple was chargedwith Montmorillonite K10 (274 g, 274 mmol) and water (54.2 mL). Themixture was vigorously stirred for ˜10 min, when a free flowing powderwas obtained. At this time DCM (1.26 mL) was added followed by Oxone(421 g, 685 mmol). The reaction temperature dropped to 15° C. A solutionof2-((1S)-1-(5-chloro-3-(2-(methylthio)phenyl)quinoxalin-2-yl)ethyl)isoindoline-1,3-dione(126 g, 274 mmol) in DCM (630 mL) was added at rt (18-20° C.) to themontmorillonite/oxone suspension through an addition funnel and theinternal temperature was controlled below 21° C. by a water/ice bath.The reaction was stirred at rt (19-21° C.) and monitored by LC. It wasdeemed completion after 96 h. The reaction mixture was filtered and thesolids were washed with DCM (2×250 mL). The combined organic solutionwas washed with 10 wt % aq. solution of Na₂SO₃ (2×250 mL), concentratedand dried to afford the product as a light yellow solid 110 g with 97.6%LC purity and 82% yield. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.14-8.29 (m,1H), 7.64-8.14 (m, 8H), 7.52 (dd, J=5.5, 3.0 Hz, 1H), 7.21-7.38 (m, 1H),5.59-5.94 (m, 1H), 3.10-3.27 (m, 3H), 1.66-1.91 (m, 3H) Mass Spectrum(ESI) m/z=492.0 (M+1).

(S)-1-(5-chloro-3-(2-(methylsulfonyl)phenyl)quinoxalin-2-yl)ethanamine

A 5 L three-neck round bottom flask equipped with an overhead stirrer,thermocouple and reflux condenser with a nitrogen inlet was charged with(S)-2-(1-(5-chloro-3-(2-(methylsulfonyl)phenyl)quinoxalin-2-yl)ethyl)isoindoline-1,3-dione(193 g, 392 mmol), water (1351 mL) and hydrazine, 35 wt. % solution inwater (711 mL, 785 mmol). The resulting white slurry was vigorouslystirred at 65° C. under nitrogen for 6 h and then it was allowed to coolto rt overnight. After this time LC-MS analysis showed completeconversion to the desired product. The white solid was isolated byfiltration, washed with water and dried on the glass filter under astream of nitrogen for 4 h to give(S)-1-(5-chloro-3-(2-(methylsulfonyl)phenyl)quinoxalin-2-yl)ethanamine(129.7 g). Mass Spectrum (ESI) m/z=362.0 (M+1).

(S)-4-amino-6-((1-(5-chloro-3-(2-(methylsulfonyl)phenyl)quinoxalin-2-yl)-ethyl)amino)pyrimidine-5-carbonitrile

A 3 L three-necked round bottom flask equipped with an overhead stirrer,nitrogen inlet and thermocouple was charged with(1S)-1-(5-chloro-3-(2-(methylsulfonyl)phenyl)quinoxalin-2-yl)ethanamine(130 g, 358 mmol), 4-amino-6-chloropyrimidine-5-carbonitrile (55.4 g,358 mmol), butan-1-ol (920 mL) and N,N-diisopropylethylamine (187 mL,1.08 mol). The reaction mixture was heated to an internal temperature of95-100° C. for 6 h and then allowed to cool to rt overnight. After thistime the reaction was heated to an internal temperature of 100° C. andthen cooled to an internal temperature of 50° C. Ethyl acetate (˜1.5 L)was added at 50° C. and the reaction was allowed to cool to rt. Theresulting solution was washed with water (3×1 L), 10 wt % aq. NaHSO₄(4×1 L) and brine (1×500 mL). The separated organic layer was dried overMgSO₄, filtered and concentrated in vacuo. The resulting residue wassuspended in toluene (1 L), evaporated in vacuo and dried under highvacuum for 36 h (247 g of crude material was obtained). The crudematerial was suspended in EtOAc (2 L), filtered and washed with a 10 wt% aqueous NaHSO₄ solution (4×1 L). A white solid started to form in theEtOAc layer. A magnetic stir bar was added and the suspension wasstirred at rt overnight and at 0° C. for 2 h. The white solid wasisolated by filtration of the EtOAc and dried on a glass filter under astream of nitrogen for two days, the EtOAc filtrate (filtrate A) was setaside. The white solid (51 g) was then suspended in EtOAc (2 L) andsaturated aqueous NaHCO₃ was added (1 L). The resulting suspension wasstirred for 15 min resulting in a two layer mixture. The organic layerwas separated, dried over MgSO₄, filtered and concentrated in vacuo togive the desired product. Filtrate A was then concentrated in vacuo,adsorbed on silica and purified by MPLC (DCM/MeOH+10% NH₄OH: 100/0 to90/10) to give 92 g of desired product. The two isolated products werecombined and dissolved in EtOH (500 mL). The resulting solution wasconcentrated in vacuo. This process of dissolution in EtOH andconcentration was repeated three times (3×500 mL). The resulting solidwas ground (mortar and pestle) to a powder and dried in a vacuum oven(temperature: 90-100° C.) over P₂O₅ for 84 h. After this time thematerial was again ground, transferred to an oversized pan and driedunder high vacuum at 100-110° C. for 24 h to give(S)-4-amino-6-((1-(5-chloro-3-(2-(methylsulfonyl)phenyl)quinoxalin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile(127.9 g). ¹H NMR (DMSO-d₆) δ ppm 8.06-8.20 (m, 3H), 7.96-7.77 (m, 5H),7.64-7.65 and 6.79-6.80 (m, 1H), 7.28 (br.s., 2H), 5.42-5.45 (m, 1H),3.32 and 3.22 (s, 3H), 1.51 and 1.40 (d, J=6.6 Hz, 3H). Mass Spectrum(ESI) m/z=480.0 (M+1). Karl Fisher and GC analysis of an analyticalsample showed that the material contained 0.45 wt % of water and 0.55 wt% of EtOH.

Example 6 Preparation of2-((S)-1-(6-amino-5-cyanopyrimidin-4-ylamino)-ethyl)-3-(2-(methylsulfonyl)phenyl)quinoxaline-5-carbonitrile2-((S)-1-(1,3-dioxoisoindolin-2-yl)ethyl)-3-(2-(methylsulfonyl)phenyl)-quinoxaline-5-carbonitrile

A 5 L three-necked round-bottomed flask equipped with a condenser,nitrogen inlet, overhead stirrer and thermocouple was charged with2-((1S)-1-(5-chloro-3-(2-(methylsulfonyl)phenyl)quinoxalin-2-yl)ethyl)isoindoline-1,3-dione(400 g, 813 mmol), dicyanozinc (143 g, 1.22 mol) and 1,4-dioxane (4.0L). The solution was stirred vigorously and degassed with Ar for 1 h. Tothe solution was then added XPhos precatalyst (66.1 g, 89 mmol). Themixture was then degassed with Ar for 1 h and heated to 90° C. Thereaction was monitored by LC, and deemed complete after 8 h. Thereaction was cooled to rt (20-21° C.) and divided into two batches. Foreach batch, EtOAc (2.0 L), NaHCO₃ (400 mL) and water (400 mL) wereadded. The mixture was stirred for 10 min and a precipitate formed. Theprecipitates were filtered to afford the pure product as a white solid201.5 g. The mother liquor was washed with brine (800 mL) and theorganic layer concentrated to afford the crude product ˜400 g. The crudematerial was then slurried in EtOAc (1.2 L) for 30 min at rt. The solidwas filtered, washed with EtOAc (2×400 mL) and dried to afford theproduct as a tan solid 220 g. All the solids were combined, dried on aglass frit under mild vacuum and a stream of N₂ for 2 days to afford theproduct as white solid 378 g with 99.6% LC purity and 96% yield. ¹H NMR(400 MHz, DMSO-d₆) δ ppm 8.47-8.68 (m, 2H), 7.66-8.19 (m, 7H), 7.52 (dd,J=5.5, 3.0 Hz, 1H), 7.22-7.40 (m, 1H), 5.61-5.95 (m, 1H), 3.11-3.27 (m,3H), 1.68-1.93 (m, 3H) Mass Spectrum (ESI) m/z=483.0 (M+1).

2-((S)-1-aminoethyl)-3-(2-(methylsulfonyl)phenyl)quinoxaline-5-carbonitrile

A 5 L three-necked round-bottomed flask equipped with a nitrogen inlet,overhead stirrer and thermocouple was charged with hydrazine hydrate(381 mL, 783 mmol) and EtOH (3.0 L). To the solution was then added2-((S)-1-(1,3-dioxoisoindolin-2-yl)ethyl)-3-(2-(methylsulfonyl)phenyl)quinoxaline-5-carbonitrile(378 g, 783 mmol) in one portion and heated to 80° C. The reaction wasmonitored by LC, and deemed complete once the reaction again reached 80°C. The reaction was cooled to room temp (20-21° C.), when DCM (3.0 L)and satd. NaHCO₃ solution (600 mL) were added. After phase separation,the organic layer was washed with brine (2×600 mL), concentrated anddried under vacuum and N₂ flow for 24 h to afford the product as a lightyellow solid 257.7 g with 94.4% LC purity, (no R enantiomer detected)and 93% yield. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.45-8.54 (m, 2H), 8.18(dt, J=7.7, 1.5 Hz, 1H), 8.05 (dd, J=8.5, 7.4 Hz, 1H), 7.76-8.00 (m,3H), 3.83-4.03 (m, 1H), 3.25-3.32 (m, 3H), 1.19-1.39 (m, 3H) MassSpectrum (ESI) m/z=353.0 (M+1).

2-((S)-1-(6-amino-5-cyanopyrimidin-4-ylamino)ethyl)-3-(2-(methylsulfonyl)-phenyl)quinoxaline-5-carbonitrile

A 5 L three-necked round-bottomed flask equipped with a mechanicalstirrer, a condenser, a nitrogen gas inlet and a temperature probe wascharged with2-((S)-1-aminoethyl)-3-(2-(methylsulfonyl)phenyl)quinoxaline-5-carbonitrile(257.7 g, 731 mmol), 4-amino-6-chloropyrimidine-5-carbonitrile (120 g,775 mmol), and butan-1-ol (2.5 L). The solution was stirred at rt(19-22° C.) for 5 min and N,N-diisopropylethylamine (363 mL, 219 mmol)was added in one portion. The solution was heated to 95° C., monitoredby LC, and deemed complete after 4 h. The reaction was cooled to rt whenEtOAc (2.0 L) and water (500 mL) were added. The two layers wasseparated and the organic layer was washed with brine (500 mL),concentrated until most of the EtOAc was removed, and a precipitateformed. The precipitate was filtered and washed with a minimum amount ofMeOH. The solid was dried under vacuum for 18 h to afford a light yellowsolid, 305 g. The mother liquor was concentrated, until more productprecipitated. The solid was filtered, washed with MeOH (2×200 mL), driedunder vacuum for 24 h to afford the product as a solid (45 g). A 5 Lthree-necked round-bottomed flask equipped with a mechanical stirrer, anitrogen gas inlet and a temperature probe was charged with the combined350 g crude product and MeOH (3.5 L). The mixture was stirred at rt(19-22° C.) for 2 days. The solid was filtered, washed with MeOH (2×350mL), dried under vacuum and N₂ flow for 24 h to afford a cream coloredsolid (276.2 g) with 99.4% HPLC purity, (no R-enantiomer detected) and80% yield. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.43-8.59 (m, 2H), 8.01-8.23(m, 2H), 7.71-7.99 (m, 4H), 7.61 (d, J=7.0 Hz, 1H), 7.24 (br. s., 2H),5.33-5.68 (m, 1H), 3.19-3.33 (m, 3H), 1.34-1.55 (m, 3H) Mass Spectrum(ESI) m/z=471.1 (M+1).

Example 7 Preparation of(S)-4-amino-6-((1-(6-fluoro-3-(pyridine-2-yl)-quinoxalin-2-yl)ethyl)amino)pyrimidine-5-carbonitrile2-((4-fluoro-2-nitrophenyl)amino)butanoic acid

A mixture of 2,5-difluoronitrobenzene (119 ml, 1.10 mol),2-aminobutanoic acid (114 g, 1.10 mol), and potassium carbonate (152.2g, 1.10 mol) in dimethyl sulfoxide (410 ml, 1.10 mol) was stirred at 80°C. for 23 h. ([Note 1]: The mixture had a deep orange red color. Theinternal temperature of the mixture went up to ˜110° C. for 1 h and thenwent down to 80° C.). After 23 h, the reaction was cooled to rt andcarefully poured into water (2 L+1 L). The aqueous mixture was washedwith diethyl ether (1 L×2) to remove organic impurities. The aqueouslayer was then acidified to ˜pH 1.5 with concentrated HCl (300 mL) togenerate a yellow solid. The yellow solid was collected by filtration,washed with water (3 L), and air-dried to give the desired product, awet orange solid. The wet orange solid was recrystallized from 3 Ltoluene and stood at rt overnight to afford the desired product as anorange crystalline solid. The orange crystalline solid was filtered,washed with toluene (2 L), and dried under high vacuum at 80° C. for 4 hand then on lyophilizer overnight to afford2-(4-fluoro-2-nitrophenylamino)-butanoic acid (203.6 g, 76.4% yield) asan orange crystalline solid. The orange crystalline solid was carried onwithout further purification to the next step.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 13.30 (br. s. 1H), 8.28 (d, J=7.4 Hz,1H), 7.90 (dd, J=9.4, 2.9 Hz, 1H), 7.49-7.60 (m, 1H), 7.09 (dd, J=9.6,4.7 Hz, 1H), 4.49 (dt, J=7.2, 5.5 Hz, 1H), 1.79-2.00 (m, 2H), 0.85-0.93(m, 3H), Mass Spectrum (ESI) m/e=243.1 (M+1).

3-Ethyl-7-fluoro-3,4-dihydroquinoxalin-2(1H)-one

The heterogeneous mixture of 2-(4-fluoro-2-nitrophenylamino)butanoicacid (181.7 g, 750.0 mmol), 3 N aq. HCl (750.0 ml, 2250 mmol), andethanol (1923 ml, 750.0 mmol) in 5-L three neck round bottom flask wasadded Tin(II) chloride dihydrate (507.7 g, 2250 mmol) and the organemixture was heated at 78° C. with stirring using an overhead stirrer for4 h ([Note 1]: The orange heterogeneous mixture became an orangehomogeneous mixture and the orange color changed to a red color over ˜2hour). After 4 h, the mixture was cooled to rt and concentrated underreduced pressure to remove ethanol, and left at rt for 48 h. Theresulting yellow precipitate was collected by filtration and washed withwater (500 mL). The yellow solid was suspended in ice water (1 L) andthe mixture was adjusted ˜pH 13 with KOH (˜250 g). The heterogeneousmixture was extracted with DCM (1 L×3). The combined organic layers werewashed with water (1 L×1) and brine (1 L×1), dried over Na₂SO₄,filtered, and concentrated under reduced pressure to give3-ethyl-7-fluoro-3,4-dihydroquinoxalin-2(1H)-one (60.92 g, 41.8% yield)as a light yellow solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 10.26 (s, 1H),6.68 (dd, J=8.6, 5.5 Hz, 1H), 6.47-6.61 (m, 2H), 5.95 (d, J=1.2 Hz, 1H),3.62 (ddd, J=6.7, 5.0, 2.0 Hz, 1H), 1.50-1.72 (m, 2H), 0.92 (t, J=7.4Hz, 3H), Mass Spectrum (ESI) m/e=195.1 (M+1).

3-Ethyl-7-fluoroquinoxalin-2(1H)-one

To a homogeneous solution of3-ethyl-7-fluoro-3,4-dihydroquinoxalin-2(1H)-one (60.61 g, 312.1 mmol)and 1,4-dioxane (1350 ml) was added2,3-dichloro-5,6-dicyano-1,4-benzoquinone (75.91 g, 327.7 mmol) and themixture was stirred using an overhead stirrer at rt for 2.5 h. At thistime, the mixture was concentrated under reduced pressure to give abrown residue. The brown residue was suspended in saturated aqueousNaHCO₃ (2 L), stirred for 30 min, filtered, washed with saturatedaqueous NaHCO₃ (1 L) to give a light green solid. The light green solidwas re-suspended in saturated aqueous NaHCO₃ (500 mL), well mixed,filtered, and washed with saturated aqueous NaHCO₃ (500 mL) to give anoff-white solid. The off-white solid was suspended in water (500 mL),well mixed, filtered, washed with water (1 L), air-dried overnight, anddried under high vacuum at 22° C. for 2 h to give3-ethyl-7-fluoroquinoxalin-2(1H)-one (56.87 g, 94.8% yield) as anoff-white solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 12.36 (br. S, 1H.),7.76 (dd, J=9.0, 5.9 Hz, 1H), 7.11 (td, J=8.8, 2.7 Hz, 1H), 7.00 (dd,J=9.6, 2.7 Hz, 1H), 2.78 (q, J=7.4 Hz, 2H), 1.20 (t, J=7.4 Hz, 3H) MassSpectrum (ESI) m/e=193.0 (M+1).

3-(1-Bromoethyl)-7-fluoroquinoxalin-2(1H)-one

3-Ethyl-7-fluoroquinoxalin-2(1H)-one (20.61 g, 107.2 mmol) and1,3-dibromo-5,5-dimethylhydantoin (18.77 g, 64.34 mmol) suspended incarbon tetrachloride (1072 ml, 107.2 mmol). To the heterogeneous mixturewas added benzoyl peroxide (3.463 g, 10.72 mmol) and the mixture washeated at reflux with stirring (oil bath temperature: 80° C.) for 20 h.After 20 h, the mixture was cooled to rt. After cooling, the mixture waspoured into saturated aqueous sodium bicarbonate solution (1 L) withstirring. The precipitate was collected by filtration and washed withwater (1 L) to give 3-(1-bromoethyl)-7-fluoroquinoxalin-2(1H)-one (23.19g, 79.8% yield) as a tan solid:

¹H NMR (400 MHz, DMSO-d₆) δ ppm 12.68 (br. S, 1H.), 7.85 (dd, J=9.0, 5.9Hz, 1H), 7.19 (td, J=8.8, 2.7 Hz, 1H), 7.05 (dd, J=9.8, 2.7 Hz, 1H),5.62 (q, J=6.7 Hz, 1H), 1.99 (d, J=6.7 Hz, 3H), ˜90% pure Mass Spectrum(ESI) m/e=271.0 [M+H(⁷⁹Br)]⁺ and 273.0 [M+H (⁸¹Br)]⁺

2-(1-Bromoethyl)-3-chloro-6-fluoroquinoxaline3-Chloro-2-(1-chloroethyl)-6-fluoroquinoxaline

A heterogeneous mixture of 3-(1-bromoethyl)-7-fluoroquinoxalin-2(1H)-one(58.86 g, 217.1 mmol) and phosphorous oxychloride (198.8 ml, 2171 mmol)in a 1 L round bottom flask was stirred at 100° C. for 2 h. The mixturewas heterogeneous in the beginning of the reaction and then homogeneousblack solution over 1 h. After 2 h, the mixture was cooled to rt andconcentrated under reduced pressure. To the black residue was carefullyadded ice (˜400 ml) in portions and then water (200 ml) with stirring.The mixture was neutralized with NH₄OH (200 ml) and ice (˜200 ml) withstirring. The resulting precipitate was collected by filtration, rinsedwith water (1 L), and dried under high vacuum overnight to give2-(1-bromoethyl)-3-chloro-6-fluoroquinoxaline (58.31 g, 92.8% yield)including 3-chloro-2-(1-chloroethyl)-6-fluoroquinoxaline as a brownsolid. ¹H NMR (400 MHz, DMSO-d₆), ratio of bromoethyl analog andchloroethyl analog=2.5:1 Mass Spectrum (ESI) m/e=288.9 [M+H(⁷⁹Br)]⁺ and291.0 [M+H (⁸¹Br)]⁺

2-(1-(3-Chloro-6-fluoroquinoxalin-2-yl)ethyl)isoindoline-1,3-dione

To a mixture of 2-(1-bromoethyl)-3-chloro-6-fluoroquinoxaline (57.17 g,197.5 mmol) containing 3-chloro-2-(1-chloroethyl)-6-fluoroquinoxaline(48.39 g, 197.5 mmol) in N,N-dimethylformamide (700.0 ml, 197.5 mmol)was added potassium phthalimide (91.43 g, 493.6 mmol) at rt and themixture was stirred at rt for 1 h. After 1 h, to the mixture was addedwater (2 L). The mixture was extracted with DCM (500 mL×3). The combinedorganic layers were washed with brine (1 L×1), dried over MgSO₄,filtered, and concentrated under reduced pressure to give a red liquid.The red liquid was filtered though a plug of silica (5.5 inch diameter×5inch height), and eluented with 20% of EtOAc in hexane, then 30% ofEtOAc in hexane, and then 100% of EtOAc as eluent to give two fractions.Second fraction, desired product2-(1-(3-chloro-6-fluoroquinoxalin-2-yl)ethyl)isoindoline-1,3-dione(53.367 g, 75.97% yield) as a pink solid. ¹H NMR (400 MHz, DMSO-d₆) δppm 8.25 (dd, J=9.4, 5.9 Hz, 1H), 7.81-7.95 (m, 6H), 5.86 (q, J=6.8 Hz,1H), 1.86 (d, J=7.0 Hz, 3H). Mass Spectrum (ESI) m/e=356.0 (M+1).

2-(1-(6-Fluoro-3-(pyridin-2-yl)quinoxalin-2-yl)ethyl)isoindoline-1,3-dione

A solution of2-(1-(3-chloro-6-fluoroquinoxalin-2-yl)ethyl)isoindoline-1,3-dione(50.14 g, 140.9 mmol), 2-tri-n-butylstannylpyridine (80% pure) (76.95ml, 211.4 mmol), and tetrakis(triphenylphosphine)palladium(0) (16.29 g,14.09 mmol) in 1,4-dioxane (1175 ml, 140.9 mmol) was stirred at 110° C.for 29 h. After 29 h, the mixture was cooled to rt and concentratedunder reduced pressure to give a green syrupy solid. To the residue wasadded DCM (200 mL). The mixture was heated under reflux for 20 min andthen cooled to 0° C. with stirring. The solid was collected byfiltration and washed with EtOAc-hexane (1:5, 500 mL) to give thedesired product (52.82 g, 94.07% yield). The dark brown solid wasdissolved in CH₂Cl₂:MeOH (9:1, 400 mL, warm), filtered through a plug ofsilica gel (150 g, 8.5 cm diameter×5.5 cm height) to remove residualpalladium and washed with CH₂Cl₂:MeOH (9:1, 600 mL). The filtrate wasconcentrated under reduced pressure to give a brown solid (49.94 g,88.9% yield). The brown solid was suspended in EtOAc-hexane (1:9, 400mL) and the mixture was heated under reflux for 40 min. The mixture wascooled to rt, filtered, washed with EtOAc-hexane (1:9, 600 mL), anddried to give the desired product2-(1-(6-fluoro-3-(pyridin-2-yl)quinoxalin-2-yl)ethyl)isoindoline-1,3-dione(47.76 g, 85.06% yield) as a tan solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm8.47-8.53 (m, 1H), 8.20 (dd, J=9.4, 5.9 Hz, 1H), 7.94 (dd, J=9.4, 2.7Hz, 1H), 7.85 (td, J=8.9, 2.9 Hz, 1H), 7.64-7.80 (m, 6H), 7.30-7.37 (m,1H), 6.42 (q, J=6.9 Hz, 1H), 1.76 (d, J=7.0 Hz, 3H). Mass Spectrum (ESI)m/e=399.1 (M+1).

1-(6-fluoro-3-(pyridin-2-yl)quinoxalin-2-yl)ethanamine

To a heterogeneous mixture of2-(1-(6-fluoro-3-(pyridin-2-yl)quinoxalin-2-yl)ethyl)isoindoline-1,3-dione(47.26 g, 118.6 mmol) in ethanol (768.3 ml, 118.6 mmol) was addedhydrazine, monohydrate (28.77 ml, 593.1 mmol) and the mixture wasstirred at 95° C. for 1 h. The heterogeneous reaction mixture went intosolution after 15 min, but voluminous white ppt followed. After 1 h themixture was cooled to rt. The precipitate was broken up with a spatula,filtered and washed with EtOAc (3×250 mL portions). The filtrate wasconcentrated under reduced pressure. The residue was redissolved inEtOAc (600 mL) and water (300 mL). The organic layer was separated andthe aqueous layer was extracted with EtOAc (100 mL×3). The combinedorganic layer were dried over MgSO₄, filtered, and concentrated underreduced pressure to give1-(6-fluoro-3-(pyridin-2-yl)quinoxalin-2-yl)ethanamine (30.95 g, 97.25%yield) as a brown solid. ¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.75 (dq,J=4.7, 0.9 Hz, 1H), 8.20 (dd, J=9.0, 5.9 Hz, 1H), 7.98-8.11 (m, 2H),7.91 (dd, J=9.4, 2.7 Hz, 1H), 7.78-7.86 (m, 1H), 7.56-7.61 (m, 1H), 4.66(q, J=6.7 Hz, 1H), 2.08 (br. S, 2H), 1.35 (d, J=6.7 Hz, 3H). MassSpectrum (ESI) m/e=269.0 (M+1). [Chiral HPLC] (Chiralpak AD-H column,0.46×250 mm, 5 um) using 10% isocratic of isopropanol in hexane aseluent: two peaks at 9.350 min and at 10.678 min at 254 nm, (ChiralpakOD-H column, 0.46×250 mm, 5 um) using 10% isocratic of isopropanol inhexane as eluent: two peaks at 9.69 min and at 11.22 min at 254 nm.

(S)-1-(6-Fluoro-3-(pyridin-2-yl)quinoxalin-2-yl)ethanamine(R)-1-(6-Fluoro-3-(pyridin-2-yl)quinoxalin-2-yl)ethanamine

Chiral Separation:

The racemic mixture (30.95 g) was submitted to chiral separation usingSFC. Sample was dissolved in 800 mL MeOH(+trace TFA), 1.3 mL samplesolution, i.e. 50.3 mg each injection onto separation system. Semi-Prepsupercritical fluid chromatography (SFC) was carried out using a Thar350 SFC (outlet pressure: 117 bar, at 327 nm) with an: AS-H column,250×30 mm (5 micron) Mobile Phase: 36 g/min MeOH(0.2% DEA) plus 54 g/minCO₂ (Temp.=22° C.) After chiral separation, each fraction wasco-evaporated two times with toluene and ethanol to remove diethylamine.First Peak on AS-H column and second peak on AD-H column (10.678 min):(R)-1-(6-fluoro-3-(pyridin-2-yl)quinoxalin-2-yl)ethanamine (13.5173 g,50.4 mmol, 43.7% yield) as a brown solid. ¹H NMR (400 MHz, DMSO-d₆) δppm 8.76 (dq, J=4.7, 0.9 Hz, 1H), 8.20 (dd, J=9.0, 5.9 Hz, 1H),7.99-8.10 (m, 2H), 7.91 (dd, J=9.4, 2.7 Hz, 1H), 7.82 (td, J=8.9, 2.9Hz, 1H), 7.56-7.61 (m, 1H), 4.66 (q, J=6.7 Hz, 1H), 2.07 (s, 2H), 1.35(d, J=6.7 Hz, 3H), it contained triphenylphosphine oxide; Mass Spectrum(ESI) m/e=269.0 (M+1). [HPLC] a peak at 4.996 min, 99.56% pure at 254nm; [Chiral HPLC] (Chiralpak AD-H column, 0.46×250 mm, 5 mm) using 10%isocratic of isopropanol in hexane as eluent: a peak at 11.432 min(second-eluting enantiomer) at 254 nm; [Enantiomeric Excess Analysis]:99% ee, first-eluting enantiomer on AS-H column. Second peak on AS-Hcolumn and first peak on AD-H column (9.350 min):(S)-1-(6-fluoro-3-(pyridin-2-yl)quinoxalin-2-yl)ethanamine (12.6738 g,47.2 mmol, 40.9% yield) as a brown syrupy solid: ¹H NMR (400 MHz,DMSO-d₆) δ ppm 8.73-8.78 (m, J=4.8, 1.2, 0.9, 0.9 Hz, 1H), 8.20 (dd,J=9.4, 5.9 Hz, 1H), 7.99-8.10 (m, 2H), 7.91 (dd, J=9.4, 2.7 Hz, 1H),7.82 (td, J=8.9, 2.9 Hz, 1H), 7.58 (ddd, J=7.4, 4.9, 1.4 Hz, 1H), 4.66(q, J=6.4 Hz, 1H), 2.07 (br. s., 2H), 1.35 (d, J=6.7 Hz, 3H); MassSpectrum (ESI) m/e=269.0 (M+1). [Chiral HPLC] (Chiralpak AD-H column,0.46×250 mm, 5 mm) using 10% isocratic of isopropanol in hexane aseluent: a peak at 9.135 min (first-eluting enantiomer) at 254 nm;[Enantiomeric Excess Analysis]: 98.86% ee, second-eluting enantiomer onAS-H column. The stereochemistry was confirmed as S-isomer in the nextstep.

(S)-4-Amino-6-((1-(6-fluoro-3-(pyridin-2-yl)quinoxalin-2-yl)ethyl)amino)-pyrimidine-5-carbonitrile

A mixture of 4-amino-6-chloropyrimidine-5-carbonitrile (0.060 g, 0.39mmol), (S)-1-(6-fluoro-3-(pyridin-2-yl)quinoxalin-2-yl)ethanamine (0.105g, 0.391 mmol), and N,N-diisopropylethylamine (0.205 mL, 1.17 mmol) inbutan-1-ol (3.91 mL) was stirred at 120° C. for 3 h. After 3 h, themixture was removed from the heat and left at rt. The mixture wasconcentrated under reduced pressure to give a yellow solid. To theyellow solid was added water (30 mL). The resulting solid was filtered,washed with water (30 mL), and air-dried to give the product as a brownsolid. The brown solid was purified by column chromatography on a 40 gof Redi-Sep column using 0 to 50% gradient of CH₂Cl₂:MeOH:NH₄OH (89:9:1)in CH₂Cl₂ over 14 min and then 50% isocratic of CH₂Cl₂:MeOH:NH₄OH(89:9:1) in CH₂Cl₂ for 14 min as eluent to give a light yellow solid(0.1246 g). The light yellow solid was suspended in EtOAc-hexane (1:4),filtered, and dried to give(S)-4-amino-6-(1-(6-fluoro-3-(pyridin-2-yl)quinoxalin-2-yl)ethylamino)pyrimidine-5-carbonitrile(0.1121 g, 0.290 mmol, 74.1% yield) as a tan solid.

¹H NMR (400 MHz, DMSO-d₆) δ ppm 8.66-8.74 (m, 1H), 8.19 (dd, J=9.4, 5.9Hz, 1H), 7.99-8.09 (m, 2H), 7.95 (dd, J=9.4, 2.7 Hz, 1H), 7.80-7.89 (m,2H), 7.70 (d, J=7.4 Hz, 1H), 7.53 (ddd, J=6.9, 5.0, 1.8 Hz, 1H), 7.20(br. s., 2H), 6.09-6.21 (m, 1H), 1.54 (d, J=6.7 Hz, 3H); Mass Spectrum(ESI) m/e=387.1 (M+1). [Chiral HPLC] (Chiralpak AD-H column, 0.46×250mm, 5 mm) using 10% isocratic of isopropanol in hexane as eluent: a peakat 16.038 min at 254 nm.

Biological Assays Recombinant Expression of PI3Ks

Full length p110 subunits of PI3k α, β and δ, N-terminally labeled withpolyHis tag, were coexpressed with p85 with Baculo virus expressionvectors in sf9 insect cells. P110/p85 heterodimers were purified bysequential Ni-NTA, Q-HP, Superdex-100 chromatography. Purified α, β andδ isozymes were stored at −20° C. in 20 mM Tris, pH 8, 0.2M NaCl, 50%glycerol, 5 mM DTT, 2 mM Na cholate. Truncated PI3Kγ, residues 114-1102,N-terminally labeled with polyHis tag, was expressed with Baculo virusin Hi5 insect cells. The γ isozyme was purified by sequential Ni-NTA,Superdex-200, Q-HP chromatography. The γ isozyme was stored frozen at−80° C. in NaH₂PO₄, pH 8, 0.2M NaCl, 1% ethylene glycol, 2 mMβ-mercaptoethanol.

Alpha Beta Delta gamma 50 mM Tris pH 8 pH 7.5 pH 7.5 pH 8 MgCl2 15 mM 10mM 10 mM 15 mM Na cholate 2 mM 1 mM 0.5 mM 2 mM DTT 2 mM 1 mM 1 mM 2 mMATP 1 uM 0.5 uM 0.5 uM 1 uM PIP2 none 2.5 uM 2.5 uM none time 1 h 2 h 2h 1 h [Enzyme] 15 nM 40 nM 15 nM 50 nM

In Vitro Enzyme Assays.

Assays were performed in 25 μL with the above final concentrations ofcomponents in white polyproplyene plates (Costar 3355). Phospatidylinositol phosphoacceptor, PtdIns(4,5)P2 P4508, was from EchelonBiosciences. The ATPase activity of the alpha and gamma isozymes was notgreatly stimulated by PtdIns(4,5)P2 under these conditions and wastherefore omitted from the assay of these isozymes. Test compounds weredissolved in dimethyl sulfoxide and diluted with three-fold serialdilutions. The compound in DMSO (1 μL) was added per test well, and theinhibition relative to reactions containing no compound, with andwithout enzyme was determined. After assay incubation at rt, thereaction was stopped and residual ATP determined by addition of an equalvolume of a commercial ATP bioluminescence kit (Perkin Elmer EasyLite)according to the manufacturer's instructions, and detected using aAnalystGT luminometer.

Human B Cells Proliferation Stimulate by Anti-IgM Isolate Human B Cells:

Isolate PBMCs from Leukopac or from human fresh blood. Isolate human Bcells by using Miltenyi protocol and B cell isolation kit II. -human Bcells were Purified by using AutoMacs.column.

Activation of Human B Cells

Use 96 well Flat bottom plate, plate 50000/well purified B cells in Bcell proliferation medium (DMEM+5% FCS, 10 mM Hepes, 50 μM2-mercaptoethanol); 150 μL medium contain 250 ng/mL CD40L-LZ recombinantprotein (Amgen) and 2 μg/mL anti-Human IgM antibody (JacksonImmunoReseach Lab.#109-006-129), mixed with 50 μL B cell mediumcontaining PI3K inhibitors and incubate 72 h at 37° C. incubator. After72 h, pulse labeling B cells with 0.5-1 uCi/well ³H thymidine forovernight ˜18 h, and harvest cell using TOM harvester.

Human B Cells Proliferation Stimulate by IL-4 Isolate Human B Cells:

Isolate human PBMCs from Leukopac or from human fresh blood. Isolatehuman B cells using Miltenyi protocol-B cell isolation kit. Human Bcells were Purified by AutoMacs.column.

Activation of Human B Cells

Use 96-well flat bottom plate, plate 50000/well purified B cells in Bcell proliferation medium (DMEM+5% FCS, 50 μM 2-mercaptoethanol, 10 mMHepes). The medium (150 μL) contain 250 ng/mL CD40L-LZ recombinantprotein (Amgen) and 10 ng/mL IL-4 (R&D system #204-IL-025), mixed with50 150 μL B cell medium containing compounds and incubate 72 h at 37° C.incubator. After 72 h, pulse labeling B cells with 0.5-1 uCi/well 3Hthymidine for overnight ˜18 h, and harvest cell using TOM harvester.

Specific T Antigen (Tetanus Toxoid) Induced Human PBMC ProliferationAssays

Human PBMC are prepared from frozen stocks or they are purified fromfresh human blood using a Ficoll gradient. Use 96 well round-bottomplate and plate 2×10⁵ PBMC/well with culture medium (RPMI1640+10% FCS,50 uM 2-Mercaptoethanol, 10 mM Hepes). For IC₅₀ determinations, PI3Kinhibitors was tested from 10 μM to 0.001 μM, in half log increments andin triplicate. Tetanus toxoid, T cell specific antigen (University ofMassachusetts Lab) was added at 1 μg/mL and incubated 6 days at 37° C.incubator. Supernatants are collected after 6 days for IL2 ELISA assay,then cells are pulsed with ³H-thymidine for ˜18 h to measureproliferation.

GFP Assays for Detecting Inhibition of Class Ia and Class III PI3K

AKT1 (PKBa) is regulated by Class Ia PI3K activated by mitogenic factors(IGF-1, PDGF, insulin, thrombin, NGF, etc.). In response to mitogenicstimuli, AKT1 translocates from the cytosol to the plasma membrane

Forkhead (FKHRL1) is a substrate for AKT1. It is cytoplasmic whenphosphorylated by AKT (survival/growth). Inhibition of AKT(stasis/apoptosis)-forkhead translocation to the nucleus

FYVE domains bind to PI(3)P. the majority is generated by constitutiveaction of PI3K Class III

AKT Membrane Ruffling Assay (CHO-IR-AKT1-EGFP cells/GE Healthcare)

Wash cells with assay buffer. Treat with compounds in assay buffer 1 h.Add 10 ng/mL insulin. Fix after 10 min at room temp and image

Forkhead Translocation Assay (MDA MB468 Forkhead-DiversaGFP Cells)

Treat cells with compound in growth medium 1 h. Fix and image.

Class III PI(3)P Assay (U2OS EGFP-2XFYVE cells/GE Healthcare)

Wash cells with assay buffer. Treat with compounds in assay buffer 1 h.Fix and image.

Control for all 3 Assays is 10 uM Wortmannin:

AKT is cytoplasmic

Forkhead is nuclear

PI(3)P depleted from endosomes

Biomarker Assay: B-Cell Receptor Stimulation of CD69 or B7.2 (CD86)Expression

Heparinized human whole blood was stimulated with 10 μg/mL anti-IgD(Southern Biotech, #9030-01). 90 μL of the stimulated blood was thenaliquoted per well of a 96-well plate and treated with 10 μL of variousconcentrations of blocking compound (from 10-0.0003 μM) diluted inIMDM+10% FBS (Gibco). Samples were incubated together for 4 h (for CD69expression) to 6 h (for B7.2 expression) at 37° C. Treated blood (50 μL)was transferred to a 96-well, deep well plate (Nunc) for antibodystaining with 10 μL each of CD45-PerCP (BD Biosciences, #347464),CD19-FITC (BD Biosciences, #340719), and CD69-PE (BD Biosciences,#341652). The second 50 μL of the treated blood was transferred to asecond 96-well, deep well plate for antibody staining with 10 μL each ofCD19-FITC (BD Biosciences, #340719) and CD86-PeCy5 (BD Biosciences,#555666). All stains were performed for 15-30 min in the dark at rt. Theblood was then lysed and fixed using 450 μL of FACS lysing solution (BDBiosciences, #349202) for 15 min at rt. Samples were then washed 2× inPBS+2% FBS before FACS analysis. Samples were gated on either CD45/CD19double positive cells for CD69 staining, or CD19 positive cells for CD86staining

Gamma Counterscreen: Stimulation of Human Monocytes for Phospho-AKTExpression

A human monocyte cell line, THP-1, was maintained in RPMI+10% FBS(Gibco). One day before stimulation, cells were counted using trypanblue exclusion on a hemocytometer and suspended at a concentration of1×10⁶ cells per mL of media. 100 μL of cells plus media (1×10⁵ cells)was then aliquoted per well of 4-96-well, deep well dishes (Nunc) totest eight different compounds. Cells were rested overnight beforetreatment with various concentrations (from 10-0.0003 μM) of blockingcompound. The compound diluted in media (12 μL) was added to the cellsfor 10 min at 37° C. Human MCP-1 (12 μL, R&D Diagnostics, #279-MC) wasdiluted in media and added to each well at a final concentration of 50ng/mL. Stimulation lasted for 2 min at rt. Pre-warmed FACS PhosflowLyse/Fix buffer (1 mL of 37° C.) (BD Biosciences, #558049) was added toeach well. Plates were then incubated at 37° C. for an additional 10-15min. Plates were spun at 1500 rpm for 10 min, supernatant was aspiratedoff, and 1 mL of ice cold 90% MeOH was added to each well with vigorousshaking Plates were then incubated either overnight at −70° C. or on icefor 30 min before antibody staining Plates were spun and washed 2× inPBS+2% FBS (Gibco). Wash was aspirated and cells were suspended inremaining buffer. Rabbit pAKT (50 μL, Cell Signaling, #4058L) at 1:100,was added to each sample for 1 h at rt with shaking Cells were washedand spun at 1500 rpm for 10 min. Supernatant was aspirated and cellswere suspended in remaining buffer. Secondary antibody, goat anti-rabbitAlexa 647 (50 μL, Invitrogen, #A21245) at 1:500, was added for 30 min atrt with shaking Cells were then washed 1× in buffer and suspended in 150μL of buffer for FACS analysis. Cells need to be dispersed very well bypipetting before running on flow cytometer. Cells were run on an LSR II(Becton Dickinson) and gated on forward and side scatter to determineexpression levels of pAKT in the monocyte population.

Gamma Counterscreen Stimulation of Monocytes for Phospho-AKT Expressionin Mouse Bone Marrow

Mouse femurs were dissected from five female BALB/c mice (Charles RiverLabs.) and collected into RPMI+10% FBS media (Gibco). Mouse bone marrowwas removed by cutting the ends of the femur and by flushing with 1 mLof media using a 25 gauge needle. Bone marrow was then dispersed inmedia using a 21 gauge needle. Media volume was increased to 20 mL andcells were counted using trypan blue exclusion on a hemocytometer. Thecell suspension was then increased to 7.5×10⁶ cells per 1 mL of mediaand 100 μL (7.5×10⁵ cells) was aliquoted per well into 4-96-well, deepwell dishes (Nunc) to test eight different compounds. Cells were restedat 37° C. for 2 h before treatment with various concentrations (from10-0.0003 μM) of blocking compound. Compound diluted in media (12 μL)was added to bone marrow cells for 10 min at 37° C. Mouse MCP-1 (12 μL,R&D Diagnostics, #479-JE) was diluted in media and added to each well ata final concentration of 50 ng/mL. Stimulation lasted for 2 min at rt. 1mL of 37° C. pre-warmed FACS Phosflow Lyse/Fix buffer (BD Biosciences,#558049) was added to each well. Plates were then incubated at 37° C.for an additional 10-15 min. Plates were spun at 1500 rpm for 10 min.Supernatant was aspirated off and 1 mL of ice cold 90% MeOH was added toeach well with vigorous shaking Plates were then incubated eitherovernight at −70° C. or on ice for 30 min before antibody stainingPlates were spun and washed 2× in PBS+2% FBS (Gibco). Wash was aspiratedand cells were suspended in remaining buffer. Fc block (2 μL, BDPharmingen, #553140) was then added per well for 10 min at rt. Afterblock, 50 μL of primary antibodies diluted in buffer; CD11b-Alexa488 (BDBiosciences, #557672) at 1:50, CD64-PE (BD Biosciences, #558455) at1:50, and rabbit pAKT (Cell Signaling, #4058L) at 1:100, were added toeach sample for 1 h at RT with shaking Wash buffer was added to cellsand spun at 1500 rpm for 10 min. Supernatant was aspirated and cellswere suspended in remaining buffer. Secondary antibody; goat anti-rabbitAlexa 647 (50 μL, Invitrogen, #A21245) at 1:500, was added for 30 min atrt with shaking Cells were then washed 1× in buffer and suspended in 100μL of buffer for FACS analysis. Cells were run on an LSR II (BectonDickinson) and gated on CD11b/CD64 double positive cells to determineexpression levels of pAKT in the monocyte population.

pAKT In Vivo Assay

Vehicle and compounds are administered p.o. (0.2 mL) by gavage (OralGavage Needles Popper & Sons, New Hyde Park, N.Y.) to mice (TransgenicLine 3751, female, 10-12 wks Amgen Inc, Thousand Oaks, Calif.) 15 minprior to the injection i.v (0.2 mLs) of anti-IgM FITC (50 ug/mouse)(Jackson Immuno Research, West Grove, Pa.). After 45 min the mice aresacrificed within a CO₂ chamber. Blood is drawn via cardiac puncture(0.3 mL) (1 cc 25 g Syringes, Sherwood, St. Louis, Mo.) and transferredinto a 15 mL conical vial (Nalge/Nunc International, Denmark). Blood isimmediately fixed with 6.0 mL of BD Phosflow Lyse/Fix Buffer (BDBioscience, San Jose, Calif.), inverted 3×'s and placed in 37° C. waterbath. Half of the spleen is removed and transferred to an eppendorf tubecontaining 0.5 mL of PBS (Invitrogen Corp, Grand Island, N.Y.). Thespleen is crushed using a tissue grinder (Pellet Pestle, Kimble/Kontes,Vineland, N.J.) and immediately fixed with 6.0 mL of BD PhosflowLyse/Fix buffer, inverted 3×'s and placed in 37° C. water bath. Oncetissues have been collected the mouse is cervically-dislocated andcarcass to disposed. After 15 min, the 15 mL conical vials are removedfrom the 37° C. water bath and placed on ice until tissues are furtherprocessed. Crushed spleens are filtered through a 70 μm cell strainer(BD Bioscience, Bedford, Mass.) into another 15 mL conical vial andwashed with 9 mL of PBS. Splenocytes and blood are spun @ 2,000 rpms for10 min (cold) and buffer is aspirated. Cells are resuspended in 2.0 mLof cold (−20° C.) 90% methyl alcohol (Mallinckrodt Chemicals,Phillipsburg, N.J.). MeOH is slowly added while conical vial is rapidlyvortexed. Tissues are then stored at −20° C. until cells can be stainedfor FACS analysis.

Multi-Dose TNP Immunization

Blood was collected by retro-orbital eye bleeds from 7-8 week old BALB/cfemale mice (Charles River Labs.) at day 0 before immunization. Bloodwas allowed to clot for 30 min and spun at 10,000 rpm in serummicrotainer tubes (Becton Dickinson) for 10 min. Sera were collected,aliquoted in Matrix tubes (Matrix Tech. Corp.) and stored at −70° C.until ELISA was performed. Mice were given compound orally beforeimmunization and at subsequent time periods based on the life of themolecule. Mice were then immunized with either 50 μg of TNP-LPS(Biosearch Tech., #T-5065), 50 μg of TNP-Ficoll (Biosearch Tech.,#F-1300), or 100 μg of TNP-KLH (Biosearch Tech., #T-5060) plus 1% alum(Brenntag, #3501) in PBS. TNP-KLH plus alum solution was prepared bygently inverting the mixture 3-5 times every 10 min for 1 h beforeimmunization. On day 5, post-last treatment, mice were CO₂ sacrificedand cardiac punctured. Blood was allowed to clot for 30 min and spun at10,000 rpm in serum microtainer tubes for 10 min. Sera were collected,aliquoted in Matrix tubes, and stored at −70° C. until further analysiswas performed. TNP-specific IgG1, IgG2a, IgG3 and IgM levels in the serawere then measured via ELISA. TNP-BSA (Biosearch Tech., #T-5050) wasused to capture the TNP-specific antibodies. TNP-BSA (10 μg/mL) was usedto coat 384-well ELISA plates (Corning Costar) overnight. Plates werethen washed and blocked for 1 h using 10% BSA ELISA Block solution(KPL). After blocking, ELISA plates were washed and serasamples/standards were serially diluted and allowed to bind to theplates for 1 h. Plates were washed and Ig-HRP conjugated secondaryantibodies (goat anti-mouse IgG1, Southern Biotech #1070-05, goatanti-mouse IgG2a, Southern Biotech #1080-05, goat anti-mouse IgM,Southern Biotech #1020-05, goat anti-mouse IgG3, Southern Biotech#1100-05) were diluted at 1:5000 and incubated on the plates for 1 h.TMB peroxidase solution (SureBlue Reserve TMB from KPL) was used tovisualize the antibodies. Plates were washed and samples were allowed todevelop in the TMB solution approximately 5-20 min depending on the Iganalyzed. The reaction was stopped with 2M sulfuric acid and plates wereread at an OD of 450 nm.

PI3K d IC50 PI3K b IC50 PI3Ka IC50 PI3Kg IC50 Ex # (mM) (mM) (mM) (mM)PI3K: b/d PI3K: a/d PI3K: g/d 1 0.0030 4.7400 9.9800 1.6100 1580 3327537 2 0.0078 1.3600 7.0400 0.8040 174 903 103 3 0.0051 0.4340 3.42000.0629 85 671 12 4 0.0100 0.5220 12.0600 0.4480 52 1206 45 5 0.00260.6520 5.1600 0.6200 251 1985 238 6 0.0070 5.8000 25.0000 2.8000 8293571 400 7 0.0117 2.4900 12.7000 3.9000 213 1085 333

on target anti-IgM in vivo pAKT mu splenocytes PI3KbMDA468 FH data, ED50pAKT (Hu splen- PI3KIII, PI3KbMBMDA468 (uM) WB, Ex # ocytes PAKT) (uM)(uM) spleenocytes 1 0.0022 >10 5.97, 2.6 0.041, 0.010 2 0.0012 >10  >10, 0.811 0.36, 0.26 3 0.0015 >10 3.8000, 0.483 no data 4 0.0018 >101.9500, 0.62  no data 5 0.0050 >10  >10, >10 0.056, 0.012 60.0060 >10 >10, 11 0.13 (WB) 7 0.0104 >10 >10 no data (Hu B Cellanti-IgM)

For the treatment of PI3Kδ-mediated-diseases, such as rheumatoidarthritis, ankylosing spondylitis, osteoarthritis, psoriatic arthritis,psoriasis, inflammatory diseases, and autoimmune diseases, the compoundsof the present invention may be administered orally, parentally, byinhalation spray, rectally, or topically in dosage unit formulationscontaining conventional pharmaceutically acceptable carriers, adjuvants,and vehicles. The term parenteral as used herein includes, subcutaneous,intravenous, intramuscular, intrasternal, infusion techniques orintraperitoneally.

Treatment of diseases and disorders herein is intended to also includethe prophylactic administration of a compound of the invention, apharmaceutical salt thereof, or a pharmaceutical composition of eitherto a subject (i.e., an animal, preferably a mammal, most preferably ahuman) believed to be in need of preventative treatment, such as, forexample, rheumatoid arthritis, ankylosing spondylitis, osteoarthritis,psoriatic arthritis, psoriasis, inflammatory diseases, and autoimmunediseases and the like.

The dosage regimen for treating PI3Kδ-mediated diseases, cancer, and/orhyperglycemia with the compounds of this invention and/or compositionsof this invention is based on a variety of factors, including the typeof disease, the age, weight, sex, medical condition of the patient, theseverity of the condition, the route of administration, and theparticular compound employed. Thus, the dosage regimen may vary widely,but can be determined routinely using standard methods. Dosage levels ofthe order from about 0.01 mg to 30 mg per kilogram of body weight perday, preferably from about 0.1 mg to 10 mg/kg, more preferably fromabout 0.25 mg to 1 mg/kg are useful for all methods of use disclosedherein.

The pharmaceutically active compounds of this invention can be processedin accordance with conventional methods of pharmacy to produce medicinalagents for administration to patients, including humans and othermammals.

For oral administration, the pharmaceutical composition may be in theform of, for example, a capsule, a tablet, a suspension, or liquid. Thepharmaceutical composition is preferably made in the form of a dosageunit containing a given amount of the active ingredient. For example,these may contain an amount of active ingredient from about 1 to 2000mg, preferably from about 1 to 500 mg, more preferably from about 5 to150 mg. A suitable daily dose for a human or other mammal may varywidely depending on the condition of the patient and other factors, but,once again, can be determined using routine methods.

The active ingredient may also be administered by injection as acomposition with suitable carriers including saline, dextrose, or water.The daily parenteral dosage regimen will be from about 0.1 to about 30mg/kg of total body weight, preferably from about 0.1 to about 10 mg/kg,and more preferably from about 0.25 mg to 1 mg/kg.

Injectable preparations, such as sterile injectable aq. or oleaginoussuspensions, may be formulated according to the known are using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed, including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables.

Suppositories for rectal administration of the drug can be prepared bymixing the drug with a suitable non-irritating excipient such as cocoabutter and polyethylene glycols that are solid at ordinary temperaturesbut liquid at the rectal temperature and will therefore melt in therectum and release the drug.

A suitable topical dose of active ingredient of a compound of theinvention is 0.1 mg to 150 mg administered one to four, preferably oneor two times daily. For topical administration, the active ingredientmay comprise from 0.001% to 10% w/w, e.g., from 1% to 2% by weight ofthe formulation, although it may comprise as much as 10% w/w, butpreferably not more than 5% w/w, and more preferably from 0.1% to 1% ofthe formulation.

Formulations suitable for topical administration include liquid orsemi-liquid preparations suitable for penetration through the skin(e.g., liniments, lotions, ointments, creams, or pastes) and dropssuitable for administration to the eye, ear, or nose.

For administration, the compounds of this invention are ordinarilycombined with one or more adjuvants appropriate for the indicated routeof administration. The compounds may be admixed with lactose, sucrose,starch powder, cellulose esters of alkanoic acids, stearic acid, talc,magnesium stearate, magnesium oxide, sodium and calcium salts ofphosphoric and sulfuric acids, acacia, gelatin, sodium alginate,polyvinyl-pyrrolidine, and/or polyvinyl alcohol, and tableted orencapsulated for conventional administration. Alternatively, thecompounds of this invention may be dissolved in saline, water,polyethylene glycol, propylene glycol, ethanol, corn oil, peanut oil,cottonseed oil, sesame oil, tragacanth gum, and/or various buffers.Other adjuvants and modes of administration are well known in thepharmaceutical art. The carrier or diluent may include time delaymaterial, such as glyceryl monostearate or glyceryl distearate alone orwith a wax, or other materials well known in the art.

The pharmaceutical compositions may be made up in a solid form(including granules, powders or suppositories) or in a liquid form(e.g., solutions, suspensions, or emulsions). The pharmaceuticalcompositions may be subjected to conventional pharmaceutical operationssuch as sterilization and/or may contain conventional adjuvants, such aspreservatives, stabilizers, wetting agents, emulsifiers, buffers etc.

Solid dosage forms for oral administration may include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compound may be admixed with at least one inert diluent such assucrose, lactose, or starch. Such dosage forms may also comprise, as innormal practice, additional substances other than inert diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets, and pills, the dosage forms may also comprise buffering agents.Tablets and pills can additionally be prepared with enteric coatings.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants, such as wetting, sweetening,flavoring, and perfuming agents.

Compounds of the present invention can possess one or more asymmetriccarbon atoms and are thus capable of existing in the form of opticalisomers as well as in the form of racemic or non-racemic mixturesthereof. The optical isomers can be obtained by resolution of theracemic mixtures according to conventional processes, e.g., by formationof diastereoisomeric salts, by treatment with an optically active acidor base. Examples of appropriate acids are tartaric, diacetyltartaric,dibenzoyltartaric, ditoluoyltartaric, and camphorsulfonic acid and thenseparation of the mixture of diastereoisomers by crystallizationfollowed by liberation of the optically active bases from these salts. Adifferent process for separation of optical isomers involves the use ofa chiral chromatography column optimally chosen to maximize theseparation of the enantiomers. Still another available method involvessynthesis of covalent diastereoisomeric molecules by reacting compoundsof the invention with an optically pure acid in an activated form or anoptically pure isocyanate. The synthesized diastereoisomers can beseparated by conventional means such as chromatography, distillation,crystallization or sublimation, and then hydrolyzed to deliver theenantiomerically pure compound. The optically active compounds of theinvention can likewise be obtained by using active starting materials.These isomers may be in the form of a free acid, a free base, an esteror a salt.

Likewise, the compounds of this invention may exist as isomers, that iscompounds of the same molecular formula but in which the atoms, relativeto one another, are arranged differently. In particular, the alkylenesubstituents of the compounds of this invention, are normally andpreferably arranged and inserted into the molecules as indicated in thedefinitions for each of these groups, being read from left to right.However, in certain cases, one skilled in the art will appreciate thatit is possible to prepare compounds of this invention in which thesesubstituents are reversed in orientation relative to the other atoms inthe molecule. That is, the substituent to be inserted may be the same asthat noted above except that it is inserted into the molecule in thereverse orientation. One skilled in the art will appreciate that theseisomeric forms of the compounds of this invention are to be construed asencompassed within the scope of the present invention.

The compounds of the present invention can be used in the form of saltsderived from inorganic or organic acids. The salts include, but are notlimited to, the following: acetate, adipate, alginate, citrate,aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate,camphorsulfonate, digluconate, cyclopentanepropionate, dodecylsulfate,ethanesulfonate, glucoheptanoate, glycerophosphate, hemisulfate,heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methansulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, palmoate,pectinate, persulfate, 2-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate, mesylate, andundecanoate. Also, the basic nitrogen-containing groups can bequaternized with such agents as lower alkyl halides, such as methyl,ethyl, propyl, and butyl chloride, bromides and iodides; dialkylsulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates, longchain halides such as decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides, aralkyl halides like benzyl and phenethylbromides, and others. Water or oil-soluble or dispersible products arethereby obtained.

Examples of acids that may be employed to from pharmaceuticallyacceptable acid addition salts include such inorganic acids ashydrochloric acid, sulfuric acid and phosphoric acid and such organicacids as oxalic acid, maleic acid, succinic acid and citric acid. Otherexamples include salts with alkali metals or alkaline earth metals, suchas sodium, potassium, calcium or magnesium or with organic bases.

Also encompassed in the scope of the present invention arepharmaceutically acceptable esters of a carboxylic acid or hydroxylcontaining group, including a metabolically labile ester or a prodrugform of a compound of this invention. A metabolically labile ester isone which may produce, for example, an increase in blood levels andprolong the efficacy of the corresponding non-esterified form of thecompound. A prodrug form is one which is not in an active form of themolecule as administered but which becomes therapeutically active aftersome in vivo activity or biotransformation, such as metabolism, forexample, enzymatic or hydrolytic cleavage. For a general discussion ofprodrugs involving esters see Svensson and Tunek Drug Metabolism Reviews165 (1988) and Bundgaard Design of Prodrugs, Elsevier (1985). Examplesof a masked carboxylate anion include a variety of esters, such as alkyl(for example, methyl, ethyl), cycloalkyl (for example, cyclohexyl),aralkyl (for example, benzyl, p-methoxybenzyl), andalkylcarbonyloxyalkyl (for example, pivaloyloxymethyl). Amines have beenmasked as arylcarbonyloxymethyl substituted derivatives which arecleaved by esterases in vivo releasing the free drug and formaldehyde(Bungaard J. Med. Chem. 2503 (1989)). Also, drugs containing an acidicNH group, such as imidazole, imide, indole and the like, have beenmasked with N-acyloxymethyl groups (Bundgaard Design of Prodrugs,Elsevier (1985)). Hydroxy groups have been masked as esters and ethers.EP 039,051 (Sloan and Little, Apr. 11, 1981) discloses Mannich-basehydroxamic acid prodrugs, their preparation and use. Esters of acompound of this invention, may include, for example, the methyl, ethyl,propyl, and butyl esters, as well as other suitable esters formedbetween an acidic moiety and a hydroxyl containing moiety. Metabolicallylabile esters, may include, for example, methoxymethyl, ethoxymethyl,iso-propoxymethyl, α-methoxyethyl, groups such asα-((C₁-C₄)-alkyloxy)ethyl, for example, methoxyethyl, ethoxyethyl,propoxyethyl, iso-propoxyethyl, etc.; 2-oxo-1,3-dioxolen-4-ylmethylgroups, such as 5-methyl-2-oxo-1,3,dioxolen-4-ylmethyl, etc.; C₁-C₃alkylthiomethyl groups, for example, methylthiomethyl, ethylthiomethyl,isopropylthiomethyl, etc.; acyloxymethyl groups, for example,pivaloyloxymethyl, α-acetoxymethyl, etc.; ethoxycarbonyl-1-methyl; orα-acyloxy-α-substituted methyl groups, for example α-acetoxyethyl.

Further, the compounds of the invention may exist as crystalline solidswhich can be crystallized from common solvents such as ethanol,N,N-dimethyl-formamide, water, or the like. Thus, crystalline forms ofthe compounds of the invention may exist as polymorphs, solvates and/orhydrates of the parent compounds or their pharmaceutically acceptablesalts. All of such forms likewise are to be construed as falling withinthe scope of the invention.

While the compounds of the invention can be administered as the soleactive pharmaceutical agent, they can also be used in combination withone or more compounds of the invention or other agents. Whenadministered as a combination, the therapeutic agents can be formulatedas separate compositions that are given at the same time or differenttimes, or the therapeutic agents can be given as a single composition.

The foregoing is merely illustrative of the invention and is notintended to limit the invention to the disclosed compounds. Variationsand changes which are obvious to one skilled in the art are intended tobe within the scope and nature of the invention which are defined in theappended claims.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

We claim:
 1. A compound selected from

or any pharmaceutically-acceptable salt thereof.
 2. A compound accordingto claim 1 having the structure

or any pharmaceutically-acceptable salt thereof.
 3. A compound accordingto claim 1 having the structure

or any pharmaceutically-acceptable salt thereof.
 4. A compound accordingto claim 1 having the structure

or any pharmaceutically-acceptable salt thereof.
 5. A compound accordingto claim 1 having the structure

or any pharmaceutically-acceptable salt thereof.
 6. A compound accordingto claim 1 having the structure

or any pharmaceutically-acceptable salt thereof.
 7. A compound accordingto claim 1 having the structure

or any pharmaceutically-acceptable salt thereof.
 8. A compound accordingto claim 1 having the structure

or any pharmaceutically-acceptable salt thereof.
 9. A pharmaceuticalcomposition comprising a therapeutically-effective amount of a compoundaccording to claim 2; and a pharmaceutically-acceptable excipient orcarrier.
 10. A pharmaceutical composition comprising atherapeutically-effective amount of a compound according to claim 3; anda pharmaceutically-acceptable excipient or carrier.
 11. A pharmaceuticalcomposition comprising a therapeutically-effective amount of a compoundaccording to claim 4; and a pharmaceutically-acceptable excipient orcarrier.
 12. A pharmaceutical composition comprising atherapeutically-effective amount of a compound according to claim 5; anda pharmaceutically-acceptable excipient or carrier.
 13. A pharmaceuticalcomposition comprising a therapeutically-effective amount of a compoundaccording to claim 6; and a pharmaceutically-acceptable excipient orcarrier.
 14. A pharmaceutical composition comprising atherapeutically-effective amount of a compound according to claim 7; anda pharmaceutically-acceptable excipient or carrier.
 15. A pharmaceuticalcomposition comprising a therapeutically-effective amount of a compoundaccording to claim 8; and a pharmaceutically-acceptable excipient orcarrier.
 16. A method of treating a condition in a mammal in needthereof, selected from of rheumatoid arthritis, ankylosing spondylitis,osteoarthritis, psoriatic arthritis, psoriasis, inflammatory diseasesand autoimmune diseases, inflammatory bowel disorders, inflammatory eyedisorders, inflammatory or unstable bladder disorders, skin complaintswith inflammatory components, chronic inflammatory conditions,autoimmune diseases, systemic lupus erythematosis (SLE), myesteniagravis, rheumatoid arthritis, acute disseminated encephalomyelitis,idiopathic thrombocytopenic purpura, multiples sclerosis, Sjoegren'ssyndrome and autoimmune hemolytic anemia, allergic conditions andhypersensitivity, comprising the step of administering atherapeutically-effective amount of compound according to claim
 2. 17. Amethod of treating a condition in a mammal in need thereof, selectedfrom of rheumatoid arthritis, ankylosing spondylitis, osteoarthritis,psoriatic arthritis, psoriasis, inflammatory diseases and autoimmunediseases, inflammatory bowel disorders, inflammatory eye disorders,inflammatory or unstable bladder disorders, skin complaints withinflammatory components, chronic inflammatory conditions, autoimmunediseases, systemic lupus erythematosis (SLE), myestenia gravis,rheumatoid arthritis, acute disseminated encephalomyelitis, idiopathicthrombocytopenic purpura, multiples sclerosis, Sjoegren's syndrome andautoimmune hemolytic anemia, allergic conditions and hypersensitivity,comprising the step of administering a therapeutically-effective amountof compound according to claim
 3. 18. A method of treating a conditionin a mammal in need thereof, selected from of rheumatoid arthritis,ankylosing spondylitis, osteoarthritis, psoriatic arthritis, psoriasis,inflammatory diseases and autoimmune diseases, inflammatory boweldisorders, inflammatory eye disorders, inflammatory or unstable bladderdisorders, skin complaints with inflammatory components, chronicinflammatory conditions, autoimmune diseases, systemic lupuserythematosis (SLE), myestenia gravis, rheumatoid arthritis, acutedisseminated encephalomyelitis, idiopathic thrombocytopenic purpura,multiples sclerosis, Sjoegren's syndrome and autoimmune hemolyticanemia, allergic conditions and hypersensitivity, comprising the step ofadministering a therapeutically-effective amount of compound accordingto claim
 4. 19. A method of treating a condition in a mammal in needthereof, selected from of rheumatoid arthritis, ankylosing spondylitis,osteoarthritis, psoriatic arthritis, psoriasis, inflammatory diseasesand autoimmune diseases, inflammatory bowel disorders, inflammatory eyedisorders, inflammatory or unstable bladder disorders, skin complaintswith inflammatory components, chronic inflammatory conditions,autoimmune diseases, systemic lupus erythematosis (SLE), myesteniagravis, rheumatoid arthritis, acute disseminated encephalomyelitis,idiopathic thrombocytopenic purpura, multiples sclerosis, Sjoegren'ssyndrome and autoimmune hemolytic anemia, allergic conditions andhypersensitivity, comprising the step of administering atherapeutically-effective amount of compound according to claim
 5. 20. Amethod of treating a condition in a mammal in need thereof, selectedfrom of rheumatoid arthritis, ankylosing spondylitis, osteoarthritis,psoriatic arthritis, psoriasis, inflammatory diseases and autoimmunediseases, inflammatory bowel disorders, inflammatory eye disorders,inflammatory or unstable bladder disorders, skin complaints withinflammatory components, chronic inflammatory conditions, autoimmunediseases, systemic lupus erythematosis (SLE), myestenia gravis,rheumatoid arthritis, acute disseminated encephalomyelitis, idiopathicthrombocytopenic purpura, multiples sclerosis, Sjoegren's syndrome andautoimmune hemolytic anemia, allergic conditions and hypersensitivity,comprising the step of administering a therapeutically-effective amountof compound according to claim
 6. 21. A method of treating a conditionin a mammal in need thereof, selected from of rheumatoid arthritis,ankylosing spondylitis, osteoarthritis, psoriatic arthritis, psoriasis,inflammatory diseases and autoimmune diseases, inflammatory boweldisorders, inflammatory eye disorders, inflammatory or unstable bladderdisorders, skin complaints with inflammatory components, chronicinflammatory conditions, autoimmune diseases, systemic lupuserythematosis (SLE), myestenia gravis, rheumatoid arthritis, acutedisseminated encephalomyelitis, idiopathic thrombocytopenic purpura,multiples sclerosis, Sjoegren's syndrome and autoimmune hemolyticanemia, allergic conditions and hypersensitivity, comprising the step ofadministering a therapeutically-effective amount of compound accordingto claim
 7. 22. A method of treating a condition in a mammal in needthereof, selected from of rheumatoid arthritis, ankylosing spondylitis,osteoarthritis, psoriatic arthritis, psoriasis, inflammatory diseasesand autoimmune diseases, inflammatory bowel disorders, inflammatory eyedisorders, inflammatory or unstable bladder disorders, skin complaintswith inflammatory components, chronic inflammatory conditions,autoimmune diseases, systemic lupus erythematosis (SLE), myesteniagravis, rheumatoid arthritis, acute disseminated encephalomyelitis,idiopathic thrombocytopenic purpura, multiples sclerosis, Sjoegren'ssyndrome and autoimmune hemolytic anemia, allergic conditions andhypersensitivity, comprising the step of administering atherapeutically-effective amount of compound according to claim 8.